Immunostimulatory G, U-containing oligoribonucleotides

ABSTRACT

Compositions and methods relating to immunostimulatory RNA oligomers are provided. The immunostimulatory RNA molecules are believed to represent natural ligands of one or more Toll-like receptors, including Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). The compositions and methods are useful for stimulating immune activation. Methods useful for screening candidate immunostimulatory compounds are also provided.

RELATED APPLICATIONS

This application is a continuation of currently pending U.S. patentapplication Ser. No. 10/407,952, filed on Apr. 4, 2003, the entirecontents of which are incorporated herein by reference, and which claimsbenefit under 35 U.S.C. §119(e) of U.S. 60/421,966, filed Oct. 29, 2002,and U.S. 60/370,515, filed Apr. 4, 2002, the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of immunology andimmune stimulation. More particularly, the present invention relates toimmunostimulatory ribonucleic acids, homologs of said immunostimulatoryribonucleic acids, and methods of use of said immunostimulatoryribonucleic acids and homologs. Compositions and methods of theinvention are believed to be useful for inducing signaling throughToll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8).

BACKGROUND OF THE INVENTION

The immune response is conceptually divided into innate immunity andadaptive immunity. Innate immunity is believed to involve recognition ofpathogen-associated molecular patterns (PAMPs) shared in common bycertain classes of molecules expressed by infectious microorganisms orforeign macromolecules. PAMPs are believed to be recognized by patternrecognition receptors (PRRs) on certain immune cells.

Toll-like receptors (TLRs) are a family of highly conserved polypeptidesthat play a critical role in innate immunity in mammals. Currently tenfamily members, designated TLR1-TLR10, have been identified. Thecytoplasmic domains of the various TLRs are characterized by aToll-interleukin 1 (IL-1) receptor (TIR) domain. Medzhitov R et al.(1998) Mol Cell 2:253-8. Recognition of microbial invasion by TLRstriggers activation of a signaling cascade that is evolutionarilyconserved in Drosophila and mammals. The TIR domain-containing adapterprotein MyD88 has been reported to associate with TLRs and to recruitIL-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF)receptor-associated factor 6 (TRAF6) to the TLRs. The MyD88-dependentsignaling pathway is believed to lead to activation of NF-kBtranscription factors and c-Jun NH₂ terminal kinase (Jnk)mitogen-activated protein kinases (MAPKs), critical steps in immuneactivation and production of inflammatory cytokines. For a review, seeAderem A et al. (2000) Nature 406:782-87.

While a number of specific TLR ligands have been reported, ligands forsome TLRs remain to be identified. Ligands for TLR2 includepeptidoglycan and lipopeptides. Yoshimura A et al. (1999) J Immunol163:1-5; Yoshimura A et al. (1999) J Immunol 163:1-5; Aliprantis A O etal. (1999) Science 285:736-9. Viral-derived double-stranded RNA (dsRNA)and poly I:C, a synthetic analog of dsRNA, have been reported to beligands of TLR3. Alexopoulou L et al. (2001) Nature 413:732-8.Lipopolysaccharide (LPS) is a ligand for TLR4. Poltorak A et al. (1998)Science 282:2085-8; Hoshino K et al. (1999) J Immunol 162:3749-52.Bacterial flagellin is a ligand for TLR5. Hayashi F et al. (2001) Nature410:1099-1103. Peptidoglycan has been reported to be a ligand not onlyfor TLR2 but also for TLR6. Ozinsky A et al. (2000) Proc Natl Acad SciUSA 97:13766-71; Takeuchi O et al. (2001) Int Immunol 13:933-40.Bacterial DNA (CpG DNA) has been reported to be a TLR9 ligand. Hemmi Het al. (2000) Nature 408:740-5; Bauer S et al. (2001) Proc Natl Acad SciUSA 98, 9237-42. The TLR ligands listed above all include naturalligands, i.e., TLR ligands found in nature as molecules expressed byinfectious microorganisms.

The natural ligands for TLR1, TLR7, TLR8 and TLR10 are not known,although recently certain low molecular weight synthetic compounds, theimidazoquinolones imiquimod (R-837) and resiquimod (R-848), werereported to be ligands of TLR7. Hemmi H et al. (2002) Nat Immunol3:196-200.

SUMMARY OF THE INVENTION

The present invention is based in part on the novel discovery by theinventors of certain immunostimulatory RNA and RNA-like (hereinafter,simply “RNA”) molecules. The immunostimulatory RNA molecules of theinvention are believed by the inventors to require a base sequence thatincludes at least one guanine (G) and at least one uracil (U), whereinoptionally the at least one G can be a variant or homolog of G and/orthe at least one U can independently be a variant or homolog of U.Surprisingly, the immunostimulatory RNA molecules of the invention canbe either single-stranded or at least partially double-stranded. Alsosurprisingly, the immunostimulatory RNA molecules of the invention donot require a CpG motif in order to exert their immunostimulatoryeffect. Without meaning to be bound by any particular theory ormechanism, it is the belief of the inventors that the immunostimulatoryRNA molecules of the invention signal through an MyD88-dependentpathway, probably through a TLR. Also without meaning to be bound by anyparticular theory or mechanism, it is the belief of the inventors thatthe immunostimulatory RNA molecules of the invention interact with andsignal through TLR8, TLR7, or some other TLR yet to be identified.

The immunostimulatory RNA molecules of the invention are also believedby the inventors to be representative of a class of RNA molecules, foundin nature, which can induce an immune response. Without meaning to bebound by any particular theory or mechanism, it is the belief of theinventors that the corresponding class of RNA molecules found in natureis believed to be present in ribosomal RNA (rRNA), transfer RNA (tRNA),messenger RNA (mRNA), and viral RNA (vRNA). It is to be noted in thisregard that the immunostimulatory RNA molecules of the present inventioncan be as small as 5-40 nucleotides long. Such short RNA molecules falloutside the range of full length messenger RNAs described to be usefulin transfecting dendritic cells in order to induce an immune response tocancer antigens. See, e.g., Boczkowski D et al. (1996) J Exp Med184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81.

It has also been discovered according to the present invention that theimmunostimulatory RNA molecules of the invention can be advantageouslycombined with with certain agents which promote stabilization of theRNA, local clustering of the RNA molecules, and/or trafficking of theRNA molecules into the endosomal compartment of cells. In particular, ithas been discovered according to the present invention that certainlipids and/or liposomes are useful in this regard. For example, certaincationic lipids, including in particular N-[1-(2,3dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP),appear to be especially advantageous when combined with theimmunostimulatory RNA molecules of the invention. As another example,covalent conjugation of a cholesteryl moiety to the RNA, for example tothe 3′ end of the RNA, promotes the immunostimulatory effect of the RNA,even in the absence of cationic lipid.

The invention provides compositions of matter and methods related to theimmunostimulatory RNA molecules of the invention. The compositions andmethods are useful, inter alia, for activating immune cells in vivo, invitro, and ex vivo; treating infection; treating cancer; preparing apharmaceutical composition; identifying a target receptor for theimmunostimulatory RNA; and screening for and characterizing additionalimmunostimulatory compounds. Furthermore, the compositions of matter andmethods related to the immunostimulatory RNA molecules of the instantinvention can advantageously be combined with other immunostimulatorycompositions of matter and methods related to such otherimmunostimulatory compositions of matter.

In one aspect the invention provides an immunostimulatory composition.The immunostimulatory composition according to this aspect of theinvention includes an isolated RNA oligomer 5-40 nucleotides long havinga base sequence having at least one guanine (G) and at least one uracil(U), and optionally a cationic lipid. The RNA oligomer can be of naturalor non-natural origin. An RNA oligomer of natural origin can in oneembodiment be derived from prokaryotic RNA and in another embodiment canbe derived from eukaryotic RNA. In addition, the RNA oligomer of naturalorigin can include a portion of a ribosomal RNA. An RNA oligomer ofnon-natural origin can include an RNA molecule synthesized outside of acell, e.g., using chemical techniques known by those of skill in theart. In one embodiment an RNA oligomer can include a derivative of anRNA oligomer of natural origin.

In one embodiment the isolated RNA oligomer is a G,U-rich RNA as definedbelow.

In one embodiment the G,U-containing immunostimulatory RNA is anisolated RNA molecule at least 5 nucleotides long which includes a basesequence as provided by 5′-RURGY-3′, wherein R represents purine, Urepresents uracil, G represents guanine, and Y represents pyrimidine. Inone embodiment the G,U-containing immunostimulatory RNA is an isolatedRNA molecule at least 5 nucleotides long which includes a base sequenceas provided by 5′-GUAGU-3′, wherein A represents adenine. In oneembodiment the G,U-containing immunostimulatory RNA is an isolated RNAmolecule which includes a base sequence as provided by 5′-GUAGUGU-3′.

In one embodiment the G,U-containing immunostimulatory RNA is anisolated RNA molecule at least 5 nucleotides long which includes a basesequence as provided by 5′-GUUGB-3′, wherein B represents U, G, or C.

In one embodiment the G,U-containing immunostimulatory RNA is anisolated RNA molecule at least 5 nucleotides long which includes a basesequence as provided by 5′-GUGUG-3′.

In other embodiments the isolated RNA molecule can contain multiples ofany of the foregoing sequences, combinations of any of the foregoingsequences, or combinations of any of the foregoing sequences includingmultiples of any of the foregoing sequences. The multiples andcombinations can be linked directly or they can be linked indirectly,i.e, through an intervening nucleoside or sequence. In one embodimentthe intervening linking nucleoside is G; in one embodiment theintervening linking nucleoside is U.

In one embodiment the base sequence includes 5′-GUGUUUAC-3′. In oneembodiment the base sequence is 5′-GUGUUUAC-3′.

In another embodiment the the base sequence includes 5′-GUAGGCAC-3′. Inone embodiment the the base sequence is 5′-GUAGGCAC-3′.

In yet another embodiment the base sequence includes 5′-CUAGGCAC-3′. Inone embodiment the base sequence is 5′-CUAGGCAC-3′.

In still another embodiment the base sequence includes 5′-CUCGGCAC-3′.In one embodiment the base sequence is 5′-CUCGGCAC-3′.

In one embodiment the oligomer is 5-12 nucleotides long. In oneembodiment the oligomer is 8-12 nucleotides long.

Also according to this aspect of the invention, in one embodiment thebase sequence is free of CpG dinucleotide. Thus in this embodiment theimmunostimulatory RNA is not a CpG nucleic acid.

In certain embodiments according to this aspect of the invention, thebase sequence of the RNA oligomer is at least partiallyself-complementary. In one embodiment the extent of self-complementarityis at least 50 percent. The extent of self-complementarity can extend toand include 100 percent. Thus for example the base sequence of the atleast partially self-complementary RNA oligomer in various embodimentscan be at least 50 percent, at least 60 percent, at least 70 percent, atleast 80 percent, at least 90 percent, or 100 percentself-complementary. Complementary base pairs include guanine-cytosine(G-C), adenine-uracil (A-U), adenine-thymine (A-T), and guanine-uracil(G-U). G-U “wobble” basepairing, which is fairly common in ribosomal RNAand in RNA retroviruses, is somewhat weaker than traditionalWatson-Crick basepairing between G-C, A-T, or A-U. A partiallyself-complementary sequence can include one or more portions ofself-complementary sequence. In an embodiment which involves a partiallyself-complementary sequence, the RNA oligomer can include aself-complementary portion positioned at and encompassing each end ofthe oligomer.

In one embodiment according to this aspect of the invention, theoligomer is a plurality of oligomers, i.e., a plurality of RNA oligomerseach 6-40 nucleotides long having a base sequence comprising at leastone guanine (G) and at least one uracil (U). The plurality of oligomerscan, but need not, include sequences which are at least partiallycomplementary to one another. In one embodiment the plurality ofoligomers includes an oligomer having a first base sequence and anoligomer having a second base sequence, wherein the first base sequenceand the second base sequence are at least 50 percent complementary. Thusfor example the at least partially complementary base sequences invarious embodiments can be at least 50 percent, at least 60 percent, atleast 70 percent, at least 80 percent, at least 90 percent, or 100percent complementary. As described above, complementary base pairsinclude guanine-cytosine (G-C), adenine-uracil (A-U), adenine-thymine(A-T), and guanine-uracil (G-U). Partially complementary sequences caninclude one or more portions of complementary sequence. In an embodimentwhich involves partially complementary sequences, the RNA oligomers caninclude a complementary portion positioned at and encompassing at leastone end of the oligomers.

In one embodiment the oligomer is a plurality of oligomers whichincludes an oligomer having a base sequence including 5′-GUGUUUAC-3′ andan oligomer having a base sequence including 5′-GUAGGCAC-3′. In oneembodiment the oligomer is a plurality of oligomers which includes anoligomer having a base sequence 5′-GUGUUUAC-3′ and an oligomer having abase sequence 5′-GUAGGCAC-3′.

Further according to this aspect of the invention, in variousembodiments the oligomer includes a non-natural backbone linkage, amodified base, a modified sugar, or any combination of the foregoing.The non-natural backbone linkage can be a stabilized linkage, i.e., alinkage which is relatively resistant against RNAse or nucleasedegradation, compared with phosphodiester linkage. In one embodiment thenon-natural backbone linkage is a phosphorothioate linkage. The oligomercan include one non-natural backbone linkage or a plurality ofnon-natural backbone linkages, each selected independently of the rest.The modified base can be a modified G, U, A, or C, including the atleast one G and the at least one U of the base sequence according tothis aspect of the invention. In some embodiments the modified base canbe selected from 7-deazaguanosine, 8-azaguanosine, 5-methyluracil, andpseudouracil. The oligomer can include one modified base or a pluralityof modified bases, each selected independently of the rest. The modifiedsugar can be a methylated sugar, arabinose. The oligomer can include onemodified sugar or a plurality of modified sugars, each selectedindependently of the rest.

In one embodiment the cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP). DOTAP is believed to transport RNA oligomer into cells andspecifically traffic to the endosomal compartment, where it can releasethe RNA oligomer in a pH-dependent fashion. Once in the endosomalcompartment, the RNA can interact with certain intracellular Toll-likereceptor molecules (TLRs), triggering TLR-mediated signal transductionpathways involved in generating an immune response. Other agents withsimilar properties including trafficking to the endosomal compartmentcan be used in place of or in addition to DOTAP.

In one embodiment the immunostimulatory composition further includes anantigen. In one embodiment the antigen is an allergen. In one embodimentthe antigen is a cancer antigen. In one embodiment the antigen is amicrobial antigen.

Also according to this aspect of the invention, in another embodimentthe invention is a pharmaceutical composition. The pharmaceuticalcomposition includes an immunostimulatory composition of the inventionand a pharmaceutically acceptable carrier. Methods for preparing thepharmaceutical composition are also provided. Such methods entailplacing an immunostimulatory composition of the invention in contactwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition can be formulated in a unit dosage for convenience.

In another aspect the invention provides a method of activating animmune cell. The method involves contacting an immune cell with animmunostimulatory composition of the invention, described above, in aneffective amount to induce activation of the immune cell. In oneembodiment the activation of the immune cell involves secretion of acytokine by the immune cell. The cytokine in one embodiment is selectedfrom the group consisting of interleukin 6 (IL-6), interleukin 12(IL-12), an interferon (IFN), and tumor necrosis factor (TNF). In oneembodiment the activation of the immune cell includes secretion of achemokine. In one embodiment the secreted chemokine isinterferon-gamma-induced protein 10 (IP-10). In one embodiment theactivation of the immune cell includes expression of acostimulatory/accessory molecule by the immune cell. In one embodimentthe costimulatory/accessory molecule is selected from the groupconsisting of intercellular adhesion molecules (ICAMs, e.g., CD54),leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80,CD86), and CD40.

Also according to this aspect of the invention, in one embodiment theactivation of the immune cell involves activation of a MyD88-dependentsignal transduction pathway. MyD88 is believed to be an adapter moleculethat interacts with the Toll/interleukin-1 receptor (TIR) domain ofvarious Toll-like receptor (TLR) molecules and participates in signaltransduction pathways that ultimately result in activation of nuclearfactor kappa B (NF-κB). Thus in one embodiment the MyD88-dependentsignal transduction pathway is associated with a TLR. More particularly,in one embodiment the TLR is TLR8. In another embodiment the TLR isTLR7.

Also according to this aspect of the invention in one embodiment theimmune cell is a human immune cell. The immune cell in one embodiment isa myeloid dendritic cell.

In one embodiment of this aspect of the invention the contacting occursin vitro. In another embodiment the contacting occurs in vivo.

The invention in another aspect provides a method of inducing an immuneresponse in a subject. The method according to this aspect of theinvention involves administering to a subject an immunostimulatorycomposition of the invention in an effective amount to induce an immuneresponse in the subject. It is to be noted that the method according tothis aspect of the invention does not involve administration of anantigen to the subject. In one embodiment the subject is a human. In oneembodiment the subject has or is at risk of having a cancer. In oneembodiment the subject has or is at risk of having an infection with anagent selected from the group consisting of viruses, bacteria, fungi,and parasites. In a particular embodiment the subject has or is at riskof having a viral infection. It is also to be noted that the methodaccording to this aspect of the invention can be used to treat a subjectwith a suppressed capacity to mount an effective or desirable immuneresponse. For example the subject can have a suppressed immune systemdue to an infection, a cancer, an acute or chronic disease such askidney or liver insufficency, surgery, and an exposure to animmunosuppressive agent such as chemotherapy, radiation, certain drugs,or the like. In one embodiment the subject has or is at risk of havingan allergy or asthma. Such a subject can be exposed to or at risk ofexposure to an allergen that is associated with an allergic response orasthma in the subject.

In yet another aspect the invention provides a method of inducing animmune response in a subject. The method according to this aspect of theinvention involves administering an antigen to a subject, andadministering to the subject an immunostimulatory composition of theinvention in an effective amount to induce an immune response to theantigen. It is to be noted that the antigen can be administered before,after, or concurrently with the immunostimulatory composition of theinvention. In addition, both the antigen and the immunostimulatorycompound can be administered to the subject more than once.

In one embodiment according to this aspect of the invention the antigenis an allergen. In one embodiment according to this aspect of theinvention the antigen is a cancer antigen. The cancer antigen in oneembodiment can be a cancer antigen isolated from the subject. In anotherembodiment the antigen is a microbial antigen. The microbial antigen canbe an antigen of a virus, a bacterium, a fungus, or a parasite.

The invention further provides, in yet another aspect, a method ofinducing an immune response in a subject. The method according to thisaspect of the invention involves isolating dendritic cells of a subject,contacting the dendritic cells ex vivo with an immunostimulatorycomposition of the invention, contacting the dendritic cells ex vivowith an antigen, and administering the contacted dendritic cells to thesubject.

In one embodiment according to this aspect of the invention the antigenis an allergen. In one embodiment according to this aspect of theinvention the antigen is a cancer antigen. The cancer antigen in oneembodiment can be a cancer antigen isolated from the subject. In anotherembodiment the antigen is a microbial antigen. The microbial antigen canbe an antigen of a virus, a bacterium, a fungus, or a parasite.

An immune response arising from stimulation of one TLR can be modified,enhanced or amplified by stimulation of another TLR, and the combinedimmunostimulatory effect may be synergistic. For example, TLR9 isreported to respond to bacterial DNA and, more generally, CpG DNA. Animmune response arising from TLR9 contacting its natural ligand (or anyTLR9 ligand) may be modified, enhanced or amplified by also selectivelycontacting TLR7 with a TLR7 ligand, or by also selectively contactingTLR8 with a TLR8 ligand, or both. Likewise, an immune response arisingfrom TLR7 contacting a TLR7 ligand may be modified, enhanced oramplified by also selectively contacting TLR8 with a TLR8 ligand, or byalso selectively contacting TLR9 with CpG DNA (or any suitable TLR9ligand), or both. As yet another example, an immune response arisingfrom TLR8 contacting a TLR8 ligand may be modified, enhanced oramplified by also selectively contacting TLR7 with a TLR7 ligand, or byalso selectively contacting TLR9 with CpG DNA (or any suitable TLR9ligand), or both.

The present invention is based in part on the novel discovery by theinventors of what are believed to be natural ligands for TLR7 and TLR8.While naturally occurring ligands derived from microbes have beendescribed for certain TLRs, natural ligands for TLR7 and TLR8 have notpreviously been described. Certain synthetic small molecules,imidazoquinoline compounds, have been described as ligands for TLR7, butsuch compounds are to be distinguished from the natural ligands of thepresent invention. Hemmi H et al. (2002) Nat Immunol 3:196-200.

Isolated natural ligands of TLR7 and TLR8 are useful as compositionsthat can induce, enhance, and complement an immune response. The naturalligands of TLR7 and TLR8 are useful for preparation of novelcompositions that can induce, enhance, and complement an immuneresponse. In addition, the natural ligands of TLR7 and TLR8 are usefulfor selectively inducing TLR7- and TLR8-mediated signaling and forselectively inducing TLR7- and TLR8-mediated immune responses.Furthermore, the natural ligands of TLR7 and TLR8 are useful indesigning and performing screening assays for identification andselection of immunostimulatory compounds.

The present invention is also based in part on the novel discoveryaccording to the invention that human neutrophils strongly express TLR8.This observation is important because neutrophils are very often thefirst cells to engage infectious pathogens and thus to initiateresponses. It is believed that activated neutrophils secrete chemokinesand cytokines, which in turn are instrumental in recruiting dendriticcells. TLR9-expressing dendritic cells drawn to the site of theactivated neutrophils there become activated, thereby amplifying theimmune response.

The present invention is also based in part on the appreciation of thedifferential expression of various TLRs, including TLR7, TLR8, and TLR9,on various cells of the immune system. This segregation may be ofparticular significance in humans with respect to TLR7, TLR8, and TLR9.The immune response arising from stimulation of any one of these TLRsmay be enhanced or amplified by stimulation of another TLR, and thecombined immunostimulatory effect may be synergistic. For example, TLR9is reported to respond to bacterial DNA and, more generally, CpG DNA. Animmune response arising from TLR9 contacting its natural ligand (or anyTLR9 ligand) may be enhanced or amplified by also selectively contactingTLR7 with its natural ligand (or any suitable TLR7 ligand), or by alsoselectively contacting TLR8 with its natural ligand (or any suitableTLR8 ligand), or both. Likewise, an immune response arising from TLR7contacting its natural ligand (or any TLR7 ligand) may be enhanced oramplified by also selectively contacting TLR8 with its natural ligand(or any suitable TLR8 ligand), or by also selectively contacting TLR9with CpG DNA (or any suitable TLR9 ligand), or both. As yet anotherexample, an immune response arising from TLR8 contacting its naturalligand (or any TLR8 ligand) may be enhanced or amplified by alsoselectively contacting TLR7 with its natural ligand (or any suitableTLR7 ligand), or by also selectively contacting TLR9 with CpG DNA (orany suitable TLR9 ligand), or both.

In a further aspect the invention provides a composition including aneffective amount of a ligand for TLR8 to induce TLR8 signaling and aneffective amount of a ligand for a second TLR selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR9 and TLR10to induce signaling by the second TLR. In one embodiment the second TLRis TLR3. In one embodiment the second TLR is TLR7. In one embodiment thesecond TLR is TLR9. In one embodiment the ligand for TLR8 and the ligandfor the second TLR are linked. In yet another embodiment the compositionfurther includes a pharmaceutically acceptable carrier.

In another aspect the invention provides a composition including aneffective amount of a ligand for TLR7 to induce TLR7 signaling and aneffective amount of a ligand for a second TLR selected from the groupconsisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8, TLR9, and TLR10to induce signaling by the second TLR. In one embodiment the second TLRis TLR3. In one embodiment the second TLR is TLR8. In one embodiment thesecond TLR is TLR9. In one embodiment the ligand for TLR7 and the ligandfor the second TLR are linked. In yet another embodiment the compositionfurther includes a pharmaceutically acceptable carrier.

In a further aspect the invention provides a composition including aDNA:RNA conjugate, wherein DNA of the conjugate includes animmunostimulatory motif effective for stimulating TLR9 signaling andwherein RNA of the conjugate includes RNA effective for stimulatingsignaling by TLR3, TLR7, TLR8, or any combination thereof. In oneembodiment the immunostimulatory motif effective for stimulating TLR9signaling is a CpG motif. In another embodiment the immunostimulatorymotif effective for stimulating TLR9 signaling is poly-dT. In yetanother embodiment the immunostimulatory motif effective for stimulatingTLR9 signaling is poly-dG. In one embodiment the conjugate includes achimeric DNA:RNA backbone. In one embodiment the chimeric backboneincludes a cleavage site between the DNA and the RNA. In one embodimentthe conjugate includes a double-stranded DNA:RNA heteroduplex. In yetanother embodiment the composition further includes a pharmaceuticallyacceptable carrier.

In another aspect the invention provides a method for stimulating TLR8signaling. The method involves contacting TLR8 with an isolated RNA inan effective amount to stimulate TLR8 signaling. In one embodiment theRNA is double-stranded RNA. In one embodiment the RNA is ribosomal RNA.In one embodiment the RNA is transfer RNA. In one embodiment the RNA ismessenger RNA. In one embodiment the RNA is viral RNA. In one embodimentthe RNA is G,U-rich RNA. In one embodiment the RNA consists essentiallyof G and U.

In yet another aspect the invention provides a method for stimulatingTLR8 signaling. The method according to this aspect involves contactingTLR8 with a mixture of nucleosides consisting essentially of G and U ina ratio between 1G:50U and 10G:1U, in an amount effective to stimulateTLR8 signaling. In one embodiment the nucleosides are ribonucleosides.In one embodiment the nucleosides comprise a mixture of ribonucleosidesand deoxyribonucleosides. In one embodiment the G is a guanosinederivative selected from the group consisting of: 8-bromoguanosine,8-oxoguanosine, 8-mercaptoguanosine, 7-allyl-8-oxoguanosine, guanosineribonucleoside vanadyl complex, inosine, and nebularine.

A further aspect of the invention provides a method for stimulating TLR8signaling. The method according to this aspect involves contacting TLR8with a mixture of ribonucleoside vanadyl complexes. In one embodimentthe mixture comprises guanosine ribonucleoside vanadyl complexes.

In another aspect the invention provides a method for stimulating TLR8signaling. The method according to this aspect involves contacting TLR8with an isolated G,U-rich oligonucleotide comprising a sequence selectedfrom the group consisting of: UUGUGG, UGGUUG, GUGUGU, and GGGUUU, in anamount effective to stimulate TLR8 signaling. In one embodiment theoligonucleotide is an oligoribonucleotide. In one embodiment theoligonucleotide is 7-50 bases long. In one embodiment theoligonucleotide is 12-24 bases long. In one embodiment theoligonucleotide has a sequence

5′-GUUGUGGUUGUGGUUGUG-3′. (SEQ ID NO:1)

The invention provides in another aspect a method for stimulating TLR8signaling. The method according to this aspect involves contacting TLR8with an at least partially double-stranded nucleic acid moleculecomprising at least one G-U base pair, in an amount effective tostimulate TLR8 signaling.

In yet another aspect the invention provides a method for supplementinga TLR8-mediated immune response. The method involves contacting TLR8with an effective amount of a TLR8 ligand to induce a TLR8-mediatedimmune response, and contacting a TLR other than TLR8 with an effectiveamount of a ligand for the TLR other than TLR8 to induce an immuneresponse mediated by the TLR other than TLR8.

In a further aspect the invention provides a method for supplementing aTLR8-mediated immune response in a subject. The method according to thisaspect involves administering to a subject in need of an immune responsean effective amount of a TLR8 ligand to induce a TLR8-mediated immuneresponse, and administering to the subject an effective amount of aligand for a TLR other than TLR8 to induce an immune response mediatedby the TLR other than TLR8. In one embodiment the TLR other than TLR8 isTLR9. In one embodiment the ligand for TLR9 is a CpG nucleic acid. Inone embodiment the CpG nucleic acid has a stabilized backbone. In oneembodiment the ligand for TLR8 and the ligand for TLR9 are a conjugate.In one embodiment the conjugate comprises a double-stranded DNA:RNAheteroduplex. In one embodiment the conjugate comprises a chimericDNA:RNA backbone. In one embodiment the chimeric backbone comprises acleavage site between the DNA and the RNA.

The invention in a further aspect provides a method for stimulating TLR7signaling. The method according to this aspect involves contacting TLR7with an isolated guanosine ribonucleoside in an effective amount tostimulate TLR7 signaling. In one embodiment the guanosine ribonucleosideis a guanosine ribonucleoside derivative selected from the groupconsisting of: 8-bromoguanosine, 8-oxoguanosine, 8-mercaptoguanosine,7-allyl-8-oxoguanosine, guanosine ribonucleoside vanadyl complex,inosine, and nebularine. In one embodiment the guanosine ribonucleosidederivative is 8-oxoguanosine. In one embodiment the guanosine nucleosideis a ribonucleoside. In one embodiment the guanosine nucleosidecomprises a mixture of ribonucleosides and deoxyribonucleosides.

In another aspect the invention further provides a method forstimulating TLR7 signaling. The method according to this aspect involvescontacting TLR7 with an isolated nucleic acid comprising a terminaloxidized or halogenized guanosine in an effective amount to stimulateTLR7 signaling. In one embodiment the oxidized or halogenized guanosineis 8-oxoguanosine.

In another aspect the invention provides a method for stimulating TLR7signaling. The method according to this aspect involves contacting TLR7with an isolated RNA in an effective amount to stimulate TLR7 signaling.In one embodiment the RNA is double-stranded RNA. In one embodiment theRNA is ribosomal RNA. In one embodiment the RNA is transfer RNA. In oneembodiment the RNA is messenger RNA. In one embodiment the RNA is viralRNA. In one embodiment the RNA is G-rich RNA. In one embodiment the RNAis part of a DNA:RNA heteroduplex. In one embodiment the RNA consistsessentially of guanosine ribonucleoside.

The invention in yet another aspect provides a method for stimulatingTLR7 signaling. The method according to this aspect involves contactingTLR7 with a mixture of nucleosides consisting essentially of G and U ina ratio between 1G:50U and 10G:1U, in an amount effective to stimulateTLR7 signaling.

Provided in yet another aspect of the invention is a method forstimulating TLR7 signaling. The method according to this aspect involvescontacting TLR7 with a mixture of ribonucleoside vanadyl complexes. Inone embodiment the mixture comprises guanosine ribonucleoside vanadylcomplexes.

In a further aspect the invention provides a method for supplementing aTLR7-mediated immune response. The method according to this aspectinvolves contacting TLR7 with an effective amount of a TLR7 ligand toinduce a TLR7-mediated immune response, and contacting a TLR other thanTLR7 with an effective amount of a ligand for the TLR other than TLR7 toinduce an immune response mediated by the TLR other than TLR7.

In yet another aspect the invention provides a method for supplementinga TLR7-mediated immune response in a subject. The method involvesadministering to a subject in need of an immune response an effectiveamount of a TLR7 ligand to induce a TLR7-mediated immune response, andadministering to the subject an effective amount of a ligand for a TLRother than TLR7 to induce an immune response mediated by the TLR otherthan TLR7. In one embodiment the TLR other than TLR7 is TLR9. In oneembodiment the ligand for TLR9 is a CpG nucleic acid. In one embodimentthe CpG nucleic acid has a stabilized backbone. In one embodiment theligand for TLR7 and the ligand for TLR9 are a conjugate. In oneembodiment the conjugate comprises a double-stranded DNA:RNAheteroduplex. In one embodiment the conjugate comprises a chimericDNA:RNA backbone. In one embodiment the chimeric backbone comprises acleavage site between the DNA and the RNA.

The invention in another aspect provides a method for screeningcandidate immunostimulatory compounds. The method according to thisaspect involves measuring a TLR8-mediated reference signal in responseto an RNA reference, measuring a TLR8-mediated test signal in responseto a candidate immunostimulatory compound, and comparing theTLR8-mediated test signal to the TLR8-mediated reference signal.

In yet another aspect the invention provides a method for screeningcandidate immunostimulatory compounds, comprising measuring aTLR8-mediated reference signal in response to an imidazoquinolinereference, measuring a TLR8-mediated test signal in response to acandidate immunostimulatory compound, and comparing the TLR8-mediatedtest signal to the TLR8-mediated reference signal.

Also provided according to yet another aspect of the invention is amethod for screening candidate immunostimulatory compounds. The methodinvolves measuring a TLR7-mediated reference signal in response to animidazoquinoline reference, measuring a TLR7-mediated test signal inresponse to a candidate immunostimulatory compound, and comparing theTLR7-mediated test signal to the TLR7-mediated reference signal.

In some embodiments the imidazoquinoline is resiquimod (R-848).

In some embodiments the imidazoquinoline is imiquimod (R-837).

In a further aspect the invention also provides a method for screeningcandidate immunostimulatory compounds. The method according to thisaspect involves measuring a TLR7-mediated reference signal in responseto a 7-allyl-8-oxoguanosine reference, measuring a TLR7-mediated testsignal in response to a candidate immunostimulatory compound, andcomparing the TLR7-mediated test signal to the TLR7-mediated referencesignal.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph depicting IL-12 p40 secretion by human peripheralblood mononuclear cells (PBMCs) in response to certain stimuli includingselected G,U-containing RNA oligonucleotides with or without DOTAP(“with Liposomes” and “without Liposomes”, respectively), as measured byspecific enzyme-linked immunosorbent assay (ELISA). The lower caseletter “s” appearing in the base sequences signifies phosphorothioatelinkage.

FIG. 2 is a bar graph depicting TNF-α secretion by human PBMCs inresponse to certain stimuli including selected G,U-containing RNAoligonucleotides with or without DOTAP (“with Liposomes” and “withoutLiposomes”, respectively), as measured by specific ELISA.

FIG. 3 is a bar graph depicting dose-dependence of IL-12 p40 secretionby human PBMCs in response to various concentrations of selectedG,U-containing RNA oligonucleotides (with DOTAP), as measured byspecific ELISA.

FIG. 4 is a bar graph depicting sequence dependence of TNF-α secretionby human PBMCs in response to various selected RNA oligonucleotidesrelated to the RNA oligonucleotide GUAGGCAC (with DOTAP), as measured byspecific ELISA.

FIG. 5 is a bar graph depicting the effect of DOTAP on IL-12 p40secretion by human PBMCs in response to various stimuli, as measured byspecific ELISA.

FIG. 6 is a quartet of bar graphs depicting IL-12 p40 secretion byvarious types of murine macrophage cells in response to a variety oftest and control immunostimulatory compounds, as measured by specificELISA. Panel A, wild type macrophages in the presence of IFN-γ; Panel B,MyD88-deficient macrophages in the presence of IFN-γ; Panel C, J774macrophage cell line; Panel D, RAW 264.7 macrophage cell line.

FIG. 7 is a pair of graphs depicting the secretion of (A) TNF-α and (B)IL-12 p40 by human PBMC upon incubation with HIV-1-derived RNAsequences, with and without DOTAP. Circles, 5′-GUAGUGUGUG-3′ (SEQ IDNO:2); Triangles, 5′-GUCUGUUGUGUG-3′ (SEQ ID NO:3). Open symbols,without DOTAP; closed symbols, with DOTAP.

FIG. 8 is a graph depicting apparent relatedness among TLRs.

FIG. 9 depicts nucleic acid binding domains in TLR7, TLR8, and TLR9.

FIG. 10 is a bar graph depicting responsiveness of human PBMC tostringent response factor (SRF).

FIG. 11 is a bar graph depicting responsiveness of human PBMC to theribonucleoside vanadyl complexes (RVCs). X denotes resiquimod.

FIG. 12 is a series of three bar graphs depicting responsiveness ofhuman TLR7 and human TLR8 to individual ribonucleosides. X denotesresiquimod.

FIG. 13 is a series of three bar graphs depicting responsiveness of TLR7and TLR8 to mixtures of two ribonucleosides.

FIG. 14 is a bar graph depicting response of human PBMC to a mixture ofthe ribonucleosides G and U.

FIG. 15 is a bar graph depicting response of human PBMC to G,U-rich RNA,but not DNA, oligonucleotides.

FIG. 16 is a bar graph depicting response of human PBMC to oxidized RNA.

FIG. 17 is a series of three bar graphs depicting human TLR7 and TLR8responses to oxidized guanosine ribonucleoside. X denotes resiquimod.

FIG. 18 is a pair of bar graphs depicting human TLR7 responses tomodified guanosine ribonucleosides.

FIG. 19 is a series of aligned gel images depicting differentialexpression of TLR1-TLR9 on human CD123+ dendritic cells (CD123+ DC),CD11c+ DC, and neutrophils.

FIG. 20 is a series of three graphs depicting the ability of short,single-stranded G,U-containing RNA oligomers to induce NF-κB in HEK-293cells stably transfected with expression plasmid for human TLR7 or humanTLR8.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates in part to the discovery by the inventors of anumber of RNA and RNA-related molecules that are effective asimmunostimulatory compounds. Identification of the immunostimulatorycompounds arose through a systematic effort aimed at identifyingnaturally occurring ligands for TLR7 and TLR8. As a result of thiseffort, it has now been discovered that RNA and RNA-like moleculescontaining guanine (G) and uracil (U), including specific sequencescontaining G and U, are immunostimulatory and appear to act through anMyD88-dependent pathway, implicating TLR involvement. Significantly,some of the RNA sequences occur in highly conserved structural featuresof 5′ untranslated regions of viral RNA that are important to viralreplication. The identified immunostimulatory RNA sequences alsocorrespond to or very nearly correspond to other RNAs, including tRNAsderived from bacteria and yeast, as well as rRNA derived from bacteriaand possibly some eukaryotes. Importantly, the immunostimulatory RNA ofthe invention includes single-stranded RNA, in addition to partially orwholly double-stranded RNA, and its effect can be abrogated by RNasetreatment. Where the RNA is at least partially double-stranded, it canin one embodiment include a stem-loop structure. As described in greaterdetail below, it has been discovered according to the invention thatsingle-stranded G,U-rich RNAs as short as 5 nucleotides long canstimulate immune cells to produce large amounts of a number of cytokinesand chemokines, including TNF-α, IL-6, IL-12, type 1 interferon (e.g.,IFN-α), and IP-10.

It has now been surprisingly discovered by the inventors that certainG,U-containing RNA molecules and their analogs, but not their DNAcounterparts, are immunostimulatory. Significantly, the G,U-containingoligoribonucleotides of the invention can be substantially smaller thanthe messenger RNAs previously described to be useful in preparingdendritic cell vaccines. See, e.g., Boczkowski D et al. (1996) J Exp Med184:465-72; Mitchell D A et al. (2000) Curr Opin Mol Ther 2:176-81.Although the G,U-containing RNA molecules of the invention can besurrogates for ribosomal RNA and/or viral RNA as found in nature, theycan be as small as 5-40 nucleotides long. As described further herein,the G,U-containing oligoribonucleotides of the invention include atleast one G and at least one U. Surprisingly, elimination of either G orU from the G,U-containing oligoribonucleotides of the inventionessentially abrogates their immunostimulatory effect. The at least one Gand at least U can be adjacent to one another, or they can be separatedby intervening nucleosides or sequence. Also significantly, theimmunostimulatory G,U-containing RNA molecules of the invention do notrequire a CpG dinucleotide.

In one aspect the invention provides an immunostimulatory composition.The immunostimulatory composition according to this aspect of theinvention includes an isolated RNA oligomer 5-40 nucleotides long havinga base sequence having at least one guanine (G) and at least one uracil(U). As will be described in greater detail further below, theimmunostimulatory RNA oligomer 5-40 nucleotides long having a basesequence having at least one guanine (G) and at least one uracil (U) isadvantageously formulated such that the RNA oligomer is stabilizedagainst degradation, concentrated in or on a particle such as aliposome, and/or targeted for delivery to the endosomal compartment ofcells. In one formulation, described in the examples below, the RNAoligomer is advantageously combined with the cationic lipid DOTAP, whichis believed to assist in trafficking the G,U-containingoligoribonucleotides into the endosomal compartment. Thus, in one aspectthe invention is an immunostimulatory composition which includes an RNAoligomer 5-40 nucleotides long having a base sequence having at leastone G and at least one U and optionally a cationic lipid.

The RNA oligomer of the invention can be of natural or non-naturalorigin. RNA as it occurs in nature is a type of nucleic acid thatgenerally refers to a linear polymer of certain ribonucleoside units,each ribonucleoside unit made up of a purine or pyrimidine base and aribose sugar, linked by internucleoside phosphodiester bonds. In thisregard “linear” is meant to describe the primary structure of RNA. RNAin general can be single-stranded or double-stranded, includingpartially double-stranded.

As used herein, “nucleoside” refers to a single sugar moiety (e.g.,ribose or deoxyribose) linked to an exchangeable organic base, which iseither a substituted pyrimidine (e.g., cytosine (C), thymidine (T) oruracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)).As described herein, the nucleoside may be a naturally occuringnucleoside, a modified nucleoside, or a synthetic (artificial)nucleoside.

The terms “nucleic acid” and “oligonucleotide” are used interchangeablyto mean multiple nucleotides (i.e., molecules comprising a sugar (e.g.,ribose or deoxyribose) linked to a phosphate group and to anexchangeable organic base, which is either a substituted pyrimidine(e.g., cytosine (C), thymidine (T) or uracil (U)) or a substitutedpurine (e.g., adenine (A) or guanine (G)). As used herein, the termsrefer to oligoribonucleotides as well as oligodeoxyribonucleotides. Theterms shall also include polynucleosides (i.e., a polynucleotide minusthe phosphate) and any other organic base-containing polymer. Nucleicacid molecules can be obtained from existing nucleic acid sources (e.g.,genomic or cDNA), but are preferably synthetic (e.g., produced bynucleic acid synthesis).

The terms nucleic acid and oligonucleotide also encompass nucleic acidsor oligonucleotides with substitutions or modifications, such as in thebases and/or sugars. For example, they include nucleic acids havingbackbone sugars which are covalently attached to low molecular weightorganic groups other than a hydroxyl group at the 3′ position and otherthan a phosphate group at the 5′ position. Thus modified nucleic acidsmay include a 2′-O-alkylated ribose group. In addition, modified nucleicacids may include sugars such as arabinose instead of ribose. Thus thenucleic acids may be heterogeneous in backbone composition therebycontaining any possible combination of polymer units linked togethersuch as peptide nucleic acids (which have amino acid backbone withnucleic acid bases). In some embodiments, the nucleic acids arehomogeneous in backbone composition. Nucleic acids also includesubstituted purines and pyrimidines such as C-5 propyne modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, and other naturally and non-naturallyoccurring nucleobases, substituted and unsubstituted aromatic moieties.Other such modifications are well known to those of skill in the art.

A natural nucleoside base can be replaced by a modified nucleoside base,wherein the modified nucleoside base is for example selected fromhypoxanthine; dihydrouracil; pseudouracil; 2-thiouracil; 4-thiouracil;5-aminouracil; 5-(C₁-C₆)-alkyluracil; 5-(C₂-C₆)-alkenyluracil;5-(C₂-C₆)-alkynyluracil; 5-(hydroxymethyl)uracil; 5-chlorouracil;5-fluorouracil; 5-bromouracil; 5-hydroxycytosine;5-(C₁-C₆)-alkylcytosine; 5-(C₂-C₆)-alkenylcytosine;5-(C₂-C₆)-alkynylcytosine; 5-chlorocytosine; 5-fluorocytosine;5-bromocytosine; N²-dimethylguanine; 2,4-diamino-purine; 8-azapurine(including, in particular, 8-azaguanine); a substituted 7-deazapurine(including, in particular, 7-deazaguanine), including7-deaza-7-substituted and/or 7-deaza-8-substituted purine; or othermodifications of a natural nucleoside bases. This list is meant to beexemplary and is not to be interpreted to be limiting.

In particular, the at least one guanine base of the immunostimulatoryG,U-containing oligoribonucleotide can be a substituted or modifiedguanine such as 7-deazaguanine; 8-azaguanine; 7-deaza-7-substitutedguanine (such as 7-deaza-7-(C2-C6)alkynylguanine); 7-deaza-8-substitutedguanine; hypoxanthine; 2,6-diaminopurine; 2-aminopurine; purine;8-substituted guanine such as 8-hydroxyguanine; and 6-thioguanine. Thislist is meant to be exemplary and is not to be interpreted to belimiting.

Also in particular, the at least one uracil base of the G,U-containingoligoribonucleotide can be a substituted or modified uracil such aspseudouracil and 5-methyluracil.

For use in the instant invention, the nucleic acids of the invention canbe synthesized de novo using any of a number of procedures well known inthe art. For example, the β-cyanoethyl phosphoramidite method (BeaucageS L et al. (1981) Tetrahedron Lett 22:1859); nucleoside H-phosphonatemethod (Garegg et al. (1986) Tetrahedron Lett 27:4051-4; Froehler et al.(1986) Nucl Acid Res 14:5399-407; Garegg et al. (1986) Tetrahedron Lett27:4055-8; Gaffney et al. (1988) Tetrahedron Lett 29:2619-22). Thesechemistries can be performed by a variety of automated nucleic acidsynthesizers available in the market. These nucleic acids are referredto as synthetic nucleic acids. Alternatively, T-rich and/or TGdinucleotides can be produced on a large scale in plasmids, (seeSambrook T et al., “Molecular Cloning: A Laboratory Manual”, Cold SpringHarbor laboratory Press, New York, 1989) and separated into smallerpieces or administered whole. Nucleic acids can be prepared fromexisting nucleic acid sequences (e.g., genomic or cDNA) using knowntechniques, such as those employing restriction enzymes, exonucleases orendonucleases. Nucleic acids prepared in this manner are referred to asisolated nucleic acid. An isolated nucleic acid generally refers to anucleic acid which is separated from components which it is normallyassociated with in nature. As an example, an isolated nucleic acid maybe one which is separated from a cell, from a nucleus, from mitochondriaor from chromatin. The term “nucleic acid” encompasses both syntheticand isolated nucleic acid.

For use in vivo, the nucleic acids may optionally be relativelyresistant to degradation (e.g., are stabilized). In some embodimentsonly specific portions of the nucleic acids may optionally bestabilized. A “stabilized nucleic acid molecule” shall mean a nucleicacid molecule that is relatively resistant to in vivo degradation (e.g.,via an exo- or endo-nuclease). Stabilization can be a function of lengthor secondary structure. Nucleic acids that are tens to hundreds of kbslong are relatively resistant to in vivo degradation. For shorternucleic acids, secondary structure can stabilize and increase theireffect. For example, if the 3′ end of an nucleic acid hasself-complementarity to an upstream region, so that it can fold back andform a sort of stem loop structure, then the nucleic acid becomesstabilized and therefore exhibits more activity.

In certain embodiments according to this aspect of the invention, thebase sequence of the RNA oligomer is at least partiallyself-complementary. A self-complementary sequence as used herein refersto a base sequence which, upon suitable alignment, may formintramolecular or, more typically, intermolecular basepairing betweenG-C, A-U, and/or G-U wobble pairs. In one embodiment the extent ofself-complementarity is at least 50 percent. For example an 8-mer thatis at least 50 percent self-complementary may have a sequence capable offorming 4, 5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Suchbasepairs may but need not necessarily involve bases located at eitherend of the self-complementary RNA oligomer. Where nucleic acidstabilization may be important to the RNA oligomers, it may beadvantageous to “clamp” together one or both ends of a double-strandednucleic acid, either by basepairing or by any other suitable means. Thedegree of self-complementarity may depend on the alignment betweenoligomers, and such alignment may or may not include single- ormultiple-nucleoside overhangs. In other embodiments, the degree ofself-complementarity is at least 60 percent, at least 70 percent, atleast 80 percent, at least 90 percent, or even 100 percent. Theforegoing notwithstanding, it should be noted that double-strandednessis not a requirement of the RNA oligomers of the invention.

Similar considerations apply to intermolecular basepairing between RNAoligonucleotides of different base sequence. Thus where a plurality ofRNA oligomers are used together, the plurality of oligomers may, butneed not, include sequences which are at least partially complementaryto one another. In one embodiment the plurality of oligomers includes anoligomer having a first base sequence and an oligomer having a secondbase sequence, wherein the first base sequence and the second basesequence are at least 50 percent complementary. For example, as betweentwo 8-mers that are at least 50 percent complementary, they may form 4,5, 6, 7, or 8 G-C, A-U, and/or G-U wobble basepairs. Such basepairs maybut need not necessarily involve bases located at either end of thecomplementary RNA oligomers. The degree of complementarity may depend onthe alignment between oligomers, and such alignment may or may notinclude single- or multiple-nucleoside overhangs. In other embodiments,the degree of complementarity is at least 60 percent, at least 70percent, at least 80 percent, at least 90 percent, or even 100 percent.

Alternatively, nucleic acid stabilization can be accomplished viaphosphate backbone modifications. Preferred stabilized nucleic acids ofthe instant invention have a modified backbone. It has been demonstratedthat modification of the nucleic acid backbone provides enhancedactivity of the nucleic acids when administered in vivo. One type ofmodified backbone is a phosphate backbone modification. Inclusion inimmunostimulatory nucleic acids of at least two phosphorothioatelinkages at the 5′ end of the oligonucleotide and multiple (preferablyfive) phosphorothioate linkages at the 3′ end, can in some circumstancesprovide maximal activity and protect the nucleic acid from degradationby intracellular exo- and endonucleases. Other modified nucleic acidsinclude phosphodiester-modified nucleic acids, combinations ofphosphodiester and phosphorothioate nucleic acids, alkylphosponate andarylphosphonate, alkylphosphorothioate and arylphosphorothioate,methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy,morpholino, and combinations thereof. Nucleic acids havingphosphorothioate linkages provide maximal activity and protect thenucleic acid from degradation by intracellular exo- and endo-nucleases.and combinations thereof. Each of these combinations and theirparticular effects on immune cells is discussed in more detail withrespect to CpG nucleic acids in issued U.S. Pat. Nos. 6,207,646 and6,239,116, the entire contents of which are hereby incorporated byreference. It is believed that these modified nucleic acids may showmore stimulatory activity due to enhanced nuclease resistance, increasedcellular uptake, increased protein binding, and/or altered intracellularlocalization.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl- and alkyl-phosphonates can be made, e.g., asdescribed in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (inwhich the charged oxygen moiety is alkylated as described in U.S. Pat.No. 5,023,243 and European Pat. No. 092,574) can be prepared byautomated solid phase synthesis using commercially available reagents.Methods for making other DNA backbone modifications and substitutionshave been described. Uhlmann E et al. (1990) Chem Rev 90:544; GoodchildJ (1990) Bioconjugate Chem 1:165.

Other stabilized nucleic acids include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

Another class of backbone modifications include2′-O-methylribonucleosides (2′-OMe). These types of substitutions aredescribed extensively in the prior art and in particular with respect totheir immunostimulating properties in Zhao et al. (1999) Bioorg Med ChemLett 9:24:3453-8. Zhao et al. describes methods of preparing 2′-OMemodifications to nucleic acids.

The immunostimulatory G,U-containing RNA oligomers of the invention aretypically about 5 to about 40 nucleotides long. Thus in certain distinctembodiments, the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long. Inone embodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In oneembodiment the G,U-containing RNA oligomer can be 5, 6, 7, 8, 9, 10, 11,or 12 nucleotides long. In one embodiment the G,U-containing RNAoligomer can be 8, 9, 10, 11, or 12 nucleotides long.

For example, RNA oligomers with the following base sequences have beendiscovered to be useful in the compositions and practice of theinvention: 5′-GUGUUUAC-3′; 5′-GUAGGCAC-3′; 5′-CUAGGCAC-3′;5′-CUCGGCAC-3′; and 5′-GUGUUUAC-3′ in combination with 5′-GUAGGCAC-3′.

Because the immunostimulatory effects of the G,U-containing RNAoligomers of the invention have been discovered to be MyD88-dependent,it is the belief of the inventors that the immunostimulatoryG,U-containing RNA oligomers of the invention may interact with at leastone TLR as a step in exerting their immunostimulatory effect. Theimmunostimulatory G,U-containing RNA oligomers of the invention may thusrepresent or mimic at least portions of natural ligands for the at leastone TLR. Such natural ligands may include ribosomal RNA, eitherprokaryotic or eukaryotic, as well as certain viral RNAs. The TLR orTLRs may be TLR8, TLR7, or some yet-to-be defined TLR. Natural ligandsfor TLR1, TLR7, TLR8, and TLR10 have not previously been described.

The immunostimulatory RNA molecules of the invention have beendiscovered to occur in nature in all types of RNA, usually inassociation with highly conserved sequence or key structural feature. Inone example, immunostimulatory RNA has been discovered to occur in thecontext of an internal ribosome entry site (IRES).

An IRES is a minimal cis-acting RNA element contained within a complexstructural feature in the 5′ untranslated region (5′ UTR) of viral RNAand other mRNAs that regulates the initiation of translation of theviral genome in a cap-independent manner. Hellen C U et al. (2001) GenesDev 15:1593-1612. Cap-independent initiation of viral RNA translationwas first observed in picornaviruses. Jackson R J et al. (1990) TrendsBiochem Sci 15:477-83; Jackson R J et al. (1995) RNA 1:985-1000.

In most eukaryotic cells, mRNA translation initiation commences withrecruitment of the cap binding complex eukaryotic initiation factor(eIF)4F, composed of eIF4E (cap binding protein), eIF4A, and eIF4G, tothe 5′ capped end of the mRNA. The 40S ribosomal subunit, carrying eIF3,and the ternary initiator complex tRNA-eIF2-GTP are then recruited tothe 5′ end of the mRNA through interaction between eIF3 and eIF4G. The40S subunit then scans the mRNA in a 5′ to 3′ direction until itencounters an appropriate start codon, whereupon the anticodon ofinitiator methionine-tRNA is engaged, the 60S subunit joins to form an80S ribosome, and translation commences.

Thus the significance of an IRES, at least in the context of a virus, isbelieved to be the ability of the IRES to confer a selective advantageto the virus over usual cap-dependent translation in the cell.

The following viruses have been reported to have IRES elements in theirgenome: all picornaviruses; bovine viral diarrhea virus; classic swinefever virus; cricket paralysis virus; encephalomyocarditis virus;foot-and-mouth disease virus; Friend murine leukemia virus gag mRNA;HCV; human immunodeficiency virus env mRNA; Kaposi's sarcoma-associatedherpesvirus; Moloney murine leukemia virus gag mRNA; Plautia staliintestine virus; poliovirus; rhinovirus; Rhopalosiphum padi virus; andRous sarcoma virus. Hellen C U et al. (2001) Genes Dev 15:1593-1612.This list is not intended to be limiting.

The viral proteins of hepatitis C virus (HCV) are translated from a 9.5kb single-stranded positive sense RNA which is flanked by 5′ and 3′UTRs. The highly conserved 5′ UTR includes an IRES present in nt 40-370.Reynolds J E et al. (1996) RNA 2:867-78. The HCV 5′ UTR is believed tohave four major structural domains (I-IV), of which domains II and IIIhave subdomains. Subdomain IIId includes a 27 nt stem-loop (nt 253-279)that on the basis of in vivo mutational studies has been reported to becritical in HCV IRES-mediated translation. Kieft J S et al. (1999) J MolBiol 292:513-29; Klinck R et al. (2000) RNA 6:1423-31. The sequence ofthe IIId 27-mer is provided by 5′-GCCGAGUAGUGUUGGGUCGCGAAAGGC-3′(SEQ IDNO:4), wherein the UUGGGU forms the terminal loop. The stem-loopstructure is reported to include a number of non-Watson-Crick basepairs, typical of other RNAs, including wobble U∘G, U∘A, G∘A, and A∘Abase pairs.

As another example, the immunostimulatory RNA sequences of the inventionhave been discovered to occur in G,U-rich sequence near the 5′ end ofthe viral RNA of human immunodeficiency virus type 1 (HIV-1) that iscrucial to efficient viral RNA packaging. Russell R S et al. (2002)Virology 303:152-63. Specifically, two key G,U-rich sequences within U5,namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′ (SEQ IDNO:3), corresponding to nt 99-108 and 112-123 of strain BH10,respectively, have been found according to the present invention to behighly immunostimulatory (see Example 11 below). It will be noted thatSEQ ID NO:2 includes both GUAGU and GUGUG, and SEQ ID NO:3 includesGUGUG.

As yet another example, the immunostimulatory RNA sequences of theinvention have been found to occur in 5S ribosomal RNA loop E of a largenumber of species of bacteria.

TLR8 and TLR7 show high sequence homology to TLR9 (FIG. 8). TLR9 is theCpG-DNA receptor and transduces immunostimulatory signals. Two DNAbinding motifs have been described in TLR9 (U.S. patent application Ser.No. 09/954,987) that are also present in TLR8 and TLR7 with somemodifications (FIG. 9). Despite this similarity, however, TLR7 and TLR8do not bind CpG-DNA.

It has been discovered according to the present invention thatguanosine, particularly guanosine in combination with uracil, andcertain guanosine-containing nucleic acids and derivatives thereof, arenatural ligands of TLR8. It has been discovered according to the presentinvention that RNA, oxidized RNA, G,U-rich nucleic acids, and at leastpartially double-stranded nucleic acid molecules having at least one G-Ubase pair are TLR8 ligands. In certain preferred embodiments involvingguanosine, guanosine derivatives, and G,U-rich nucleic acids, guanosineis the ribonucleoside. Nucleic acid molecules containing GUU, GUG, GGU,GGG, UGG, UGU, UUG, UUU, multiples and any combinations thereof arebelieved to be TLR8 ligands. In some embodiments the TLR8 ligand is aG,U-rich oligonucleotide that includes a hexamer sequence (UUGUGG)_(n),(UGGUUG)_(n), (GUGUGU)_(n), or (GGGUUU)_(n) where n is an integer from 1to 8, and preferably n is at least 3. In addition, it has also beendiscovered according to the present invention that mixtures ofribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine,guanosine, and uracil ribonucleoside vanadyl complexes), and guanosineribonucleoside vanadyl complexes alone, are TLR8 ligands. In addition,it has been discovered according the present invention that certainimidazoquinolines, including resiquimod and imiquimod, are TLR8 ligands.

It has also been discovered according to the present invention thatguanosine, and certain guanosine-containing nucleic acids andderivatives thereof, are natural ligands of TLR7. It has been discoveredaccording to the present invention that RNA, oxidized RNA, G-richnucleic acids, and at least partially double-stranded nucleic acidmolecules that are rich in G content are TLR7 ligands. In certainpreferred embodiments involving guanosine, guanosine derivatives, andG-rich nucleic acids, guanosine is the ribonucleoside. In addition, ithas also been discovered according to the present invention thatmixtures of ribonucleoside vanadyl complexes (i.e., mixtures of adenine,cytosine, guanosine, and uracil ribonucleoside vanadyl complexes), andguanosine ribonucleoside vanadyl complexes alone, are TLR7 ligands. Inaddition, it has been discovered according the present invention that7-allyl-8-oxoguanosine (loxoribine) is a TLR7 ligand.

In addition to having diverse ligands, the various TLRs are believed tobe differentially expressed in various tissues and on various types ofimmune cells. For example, human TLR7 has been reported to be expressedin placenta, lung, spleen, lymph nodes, tonsil and on plasmacytoidprecursor dendritic cells (pDCs). Chuang T-H et al. (2000) Eur CytokineNetw 11:372-8); Kadowaki N et al. (2001) J Exp Med 194:863-9. Human TLR8has been reported to be expressed in lung, peripheral blood leukocytes(PBL), placenta, spleen, lymph nodes, and on monocytes. Kadowaki N etal. (2001) J Exp Med 194:863-9; Chuang T-H et al. (2000) Eur CytokineNetw 11:372-8. Human TLR9 is reportedly expressed in spleen, lymphnodes, bone marrow, PBL, and on pDCs, B cells, and CD123+ DCs. KadowakiN et al. (2001) J Exp Med 194:863-9; Bauer S et al. (2001) Proc NatlAcad Sci USA 98:9237-42; Chuang T-H et al. (2000) Eur Cytokine Netw11:372-8.

Guanosine derivatives have previously been described as B-cell and NKcell activators, but their receptors and mechanism of action were notunderstood. Goodman M G et al. (1994) J Pharm Exp Ther 274:1552-57;Reitz A B et al. (1994) J Med Chem 37:3561-78. Such guanosinederivatives include, but are not limited to, 8-bromoguanosine,8-oxoguanosine, 8-mercaptoguanosine, and 7-allyl-8-oxoguanosine(loxoribine).

Imidazoquinolines are synthetic small molecule immune response modifiersthought to induce expression of several cytokines including interferons(e.g., IFN-α and IFN-γ), tumor necrosis factor alpha (TNF-α) and someinterleukins (e.g., IL-1, IL-6 and IL-12). Imidazoquinolines are capableof stimulating a Th1 immune response, as evidenced in part by theirability to induce increases in IgG2a levels. Imidazoquinoline agentsreportedly are also capable of inhibiting production of Th2 cytokinessuch as IL-4, IL-5, and IL-13. Some of the cytokines induced byimidazoquinolines are produced by macrophages and dendritic cells. Somespecies of imidazoquinolines have been reported to increase NK celllytic activity and to stimulate B-cell proliferation anddifferentiation, thereby inducing antibody production and secretion.

As used herein, an imidazoquinoline agent includes imidazoquinolineamines, imidazopyridine amines, 6,7-fused cycloalkylimidazopyridineamines, and 1,2 bridged imidazoquinoline amines. These compounds havebeen described in U.S. Pat. Nos. 4,689,338, 4,929,624, 5,238,944,5,266,575, 5,268,376, 5,346,905, 5,352,784, 5,389,640, 5,395,937,5,494,916, 5,482,936, 5,525,612, 6,039,969 and 6,110,929. Particularspecies of imidazoquinoline agents include4-amino-α,α-dimethyl-2-ethoxymethyl-1H-imidazo[4,5-c]quinoline-1-ethanol(resiquimod or R-848 or S-28463; PCT/US01/28764, WO 02/22125); and1-(2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-amine (imiquimod orR-837 or S-26308). Imiquimod is currently used in the topical treatmentof warts such as genital and anal warts and has also been tested in thetopical treatment of basal cell carcinoma.

Nucleotide and amino acid sequences of human and murine TLR3 are known.See, for example, GenBank Accession Nos. U88879, NM_(—)003265,NM_(—)126166, AF355152; and AAC34134, NP_(—)003256, NP_(—)569054,AAK26117. Human TLR3 is reported to be 904 amino acids long and to havea sequence provided in SEQ ID NO:20. A corresponding nucleotide sequenceis provided as SEQ ID NO:21. Murine TLR3 is reported to be 905 aminoacids long and to have a sequence as provided in SEQ ID NO:22. Acorresponding nucleotide sequence is provided as SEQ ID NO:23. TLR3polypeptide includes an extracellular domain having leucine-rich repeatregion, a transmembrane domain, and an intracellular domain thatincludes a TIR domain.

As used herein a “TLR3 polypeptide” refers to a polypeptide including afull-length TLR3 according to one of the sequences above, orthologs,allelic variants, SNPs, variants incorporating conservative amino acidsubstitutions, TLR3 fusion proteins, and functional fragments of any ofthe foregoing. Preferred embodiments include human TLR3 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the human TLR3 amino acid sequence of SEQ IDNO:20. Preferred embodiments also include murine TLR3 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the murine TLR3 amino acid sequence of SEQ IDNO:22.

As used herein “TLR3 signaling” refers to an ability of a TLR3polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, alsoreferred to herein as the TLR signal transduction pathway. Changes inTLR3 activity can be measured by assays such as those disclosed herein,including expression of genes under control of κB-sensitive promotersand enhancers. Such naturally occurring genes include the genes encodingIL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), andthe costimulatory molecules CD80 and CD86. Other genes can be placedunder the control of such regulatory elements (see below) and thus serveto report the level of TLR3 signaling. Additional nucleotide sequencecan be added to SEQ ID NO:21 or SEQ ID NO:23, preferably to the 5′ orthe 3′ end of the open reading frame of SEQ ID NO:21, to yield anucleotide sequence encoding a chimeric polypeptide that includes adetectable or reporter moiety, e.g., FLAG, luciferase (luc), greenfluorescent protein (GFP), and others known by those skilled in the art.

SEQ ID NO:20     Human TLR3 amino acidMRQTLPCIYF WGGLLPFGML CASSTTKCTV SHEVADCSHL KLTQVPDDLP TNITVLNLTH 60NQLRRLPAAN FTRYSQLTSL DVGFNTISKL EPELCQKLPM LKVLNLQHNE LSQLSDKTFA 120FCTNLTELHL MSNSIQKIKN NPFVKQKNLI TLDLSHNGLS STKLGTQVQL ENLQELLLSN 180NKIQALKSEE LDIFANSSLK KLELSSNQIK EFSPGCFHAI GRLFGLFLNN VQLGPSLTEK 240LCLELANTSI RNLSLSNSQL STTSNTTFLG LKWTNLTMLD LSYNNLNVVG NDSFAWLPQL 300EYFFLEYNNI QHLFSHSLHG LFNVRYLNLK RSFTKQSISL ASLPKIDDFS FQWLKCLEHL 360NMEDNDIPGI KSNMFTGLIN LKYLSLSNSF TSLRTLTNET FVSLAHSPLH ILNLTKMKIS 420KIESDAFSWL GHLEVLDLGL NEIGQELTGQ EWRGLENIFE IYLSYNKYLQ LTRNSFALVP 480SLQRLMLRRV ALKNVDSSPS PFQPLRNLTI LDLSNNNIAN INDDMLEGLE KLEILDLQHN 540NLARLWKHAN PGGPIYFLKG LSHLHILNLE SNGFDEIPVE VFKDLFELKI IDLGLNNLNT 600LPASVFNNQV SLKSLNLQKN LITSVEKKVF GPAFRNLTEL DMRFNPFDCT CESIAWFVNW 660INETHTNIPE LSSHYLCNTP PHYHGFPVRL FDTSSCKDSA PFELFFMINT SILLIFIFIV 720LLIHFEGWRI SFYWNVSVHR VLGFKEIDRQ TEQFEYAAYI IHAYKDKDWV WEHFSSMEKE 780DQSLKFCLEE RDFEAGVFEL EAIVNSIKRS RKIIFVITHH LLKDPLCKRF KVHHAVQQAI 840EQNLDSIILV FLEEIPDYKL NHALCLRRGM FKSHCILNWP VQKERIGAFR HKLQVALGSK 900NSVH 904 SEQ ID NO:21     Human TLR3 nucleotidecactttcgag agtgccgtct atttgccaca cacttccctg atgaaatgtc tggatttgga 60ctaaagaaaa aaggaaaggc tagcagtcat ccaacagaat catgagacag actttgcctt 120gtatctactt ttgggggggc cttttgccct ttgggatgct gtgtgcatcc tccaccacca 180agtgcactgt tagccatgaa gttgctgact gcagccacct gaagttgact caggtacccg 240atgatctacc cacaaacata acagtgttga accttaccca taatcaactc agaagattac 300cagccgccaa cttcacaagg tatagccagc taactagctt ggatgtagga tttaacacca 360tctcaaaact ggagccagaa ttgtgccaga aacttcccat gttaaaagtt ttgaacctcc 420agcacaatga gctatctcaa ctttctgata aaacctttgc cttctgcacg aatttgactg 480aactccatct catgtccaac tcaatccaga aaattaaaaa taatcccttt gtcaagcaga 540agaatttaat cacattagat ctgtctcata atggcttgtc atctacaaaa ttaggaactc 600aggttcagct ggaaaatctc caagagcttc tattatcaaa caataaaatt caagcgctaa 660aaagtgaaga actggatatc tttgccaatt catctttaaa aaaattagag ttgtcatcga 720atcaaattaa agagttttct ccagggtgtt ttcacgcaat tggaagatta tttggcctct 780ttctgaacaa tgtccagctg ggtcccagcc ttacagagaa gctatgtttg gaattagcaa 840acacaagcat tcggaatctg tctctgagta acagccagct gtccaccacc agcaatacaa 900ctttcttggg actaaagtgg acaaatctca ctatgctcga tctttcctac aacaacttaa 960atgtggttgg taacgattcc tttgcttggc ttccacaact agaatatttc ttcctagagt 1020ataataatat acagcatttg ttttctcact ctttgcacgg gcttttcaat gtgaggtacc 1080tgaatttgaa acggtctttt actaaacaaa gtatttccct tgcctcactc cccaagattg 1140atgatttttc ttttcagtgg ctaaaatgtt tggagcacct taacatggaa gataatgata 1200ttccaggcat aaaaagcaat atgttcacag gattgataaa cctgaaatac ttaagtctat 1260ccaactcctt tacaagtttg cgaactttga caaatgaaac atttgtatca cttgctcatt 1320ctcccttaca catactcaac ctaaccaaga ataaaatctc aaaaatagag agtgatgctt 1380tctcttggtt gggccaccta gaagtacttg acctgggcct taatgaaatt gggcaagaac 1440tcacaggcca ggaatggaga ggtctagaaa atattttcga aatctatctt tcctacaaca 1500agtacctgca gctgactagg aactcctttg ccttggtccc aagccttcaa cgactgatgc 1560tccgaagggt ggcccttaaa aatgtggata gctctccttc accattccag cctcttcgta 1620acttgaccat tctggatcta agcaacaaca acatagccaa cataaatgat gacatgttgg 1680agggtcttga gaaactagaa attctcgatt tgcagcataa caacttagca cggctctgga 1740aacacgcaaa ccctggtggt cccatttatt tcctaaaggg tctgtctcac ctccacatcc 1800ttaacttgga gtccaacggc tttgacgaga tcccagttga ggtcttcaag gatttatttg 1860aactaaagat catcgattta ggattgaata atttaaacac acttccagca tctgtcttta 1920ataatcaggt gtctctaaag tcattgaacc ttcagaagaa tctcataaca tccgttgaga 1980agaaggtttt cgggccagct ttcaggaacc tgactgagtt agatatgcgc tttaatccct 2040ttgattgcac gtgtgaaagt attgcctggt ttgttaattg gattaacgag acccatacca 2100acatccctga gctgtcaagc cactaccttt gcaacactcc acctcactat catgggttcc 2160cagtgagact ttttgataca tcatcttgca aagacagtgc cccctttgaa ctctttttca 2220tgatcaatac cagtatcctg ttgattttta tctttattgt acttctcatc cactttgagg 2280gctggaggat atctttttat tggaatgttt cagtacatcg agttcttggt ttcaaagaaa 2340tagacagaca gacagaacag tttgaatatg cagcatatat aattcatgcc tataaagata 2400aggattgggt ctgggaacat ttctcttcaa tggaaaagga agaccaatct ctcaaatttt 2460gtctggaaga aagggacttt gaggcgggtg tttttgaact agaagcaatt gttaacagca 2520tcaaaagaag cagaaaaatt atttttgtta taacacacca tctattaaaa gacccattat 2580gcaaaagatt caaggtacat catgcagttc aacaagctat tgaacaaaat ctggattcca 2640ttatattggt tttccttgag gagattccag attataaact gaaccatgca ctctgtttgc 2700gaagaggaat gtttaaatct cactgcatct tgaactggcc agttcagaaa gaacggatag 2760gtgcctttcg tcataaattg caagtagcac ttggatccaa aaactctgta cattaaattt 2820atttaaatat tcaattagca aaggagaaac tttctcaatt taaaaagttc tatggcaaat 2880ttaagttttc cataaaggtg ttataatttg tttattcata tttgtaaatg attatattct 2940atcacaatta catctcttct aggaaaatgt gtctccttat ttcaggccta tttttgacaa 3000ttgacttaat tttacccaaa ataaaacata taagcacgta aaaaaaaaaa aaaaaaa 3057SEQ ID NO:22     Murine TLR3 amino acidMKGCSSYLMY SFGGLLSLWI LLVSSTNQCT VRYNVADCSH LKLTHIPDDL PSNITVLNLT 60HNQLRRLPPT NFTRYSQLAI LDAGFNSISK LEPELCQILP LLKVLNLQHN ELSQISDQTF 120VFCTNLTELD SMSNSIHKIK SNPFKNQKNL IKLDLSHNGL SSTKLGTGVQ LENLQELLLA 180KNKILALRSE ELEFLGNSSL RKLDLSSNPL KEFSPGCFQT IGKLFALLLN NAQLNPHLTE 240KLCWELSNTS IQNLSLANNQ LLATSESTFS GLKWTNLTQL DLSYNNLHDV GNGSFSYLPS 300LRYLSLEYNN IQRLSPRSFY GLSNLRYLSL KRAFTKQSVS LASHPNIDDF SFQWLKYLEY 360LNMDDNNIPS TKSNTFTGLV SLKYLSLSKT FTSLQTLTNE TFVSLAHSPL LTLNLTKNHI 420SKIANGTFSW LGQLRILDLG LNEIEQKLSG QEWRGLRNIF EIYLSYNKYL QLSTSSFALV 480PSLQRLMLRR VALKNVDISP SPFRPLRNLT ILDLSNNNIA NINEDLLEGL ENLEILDFQH 540NNLARLWKRA NPGGPVNFLK GLSHLHILNL ESNGLDEIPV GVFKNLFELK SINLGLNNLN 600KLEPFIFDDQ TSLRSLNLQK NLITSVEKDV FGPPFQNLNS LDMRFNPFDC TCESISWFVN 660WINQTHTNIF ELSTHYLCNT PHHYYGFPLK LFDTSSCKDS APFELLFIIS TSMLLVFILV 720VLLIHIEGWR ISFYWNVSVH RILGFKBIDT QAEQFEYTAY IIHAHKDRDW VWEHFSPMEE 780QDQSLKFCLE ERDFEAGVLG LEAIVNSIKR SRKIIFVITH HLLKDPLCRR FKVHHAVQQA 840IEQNLDSIIL IFLQNIPDYK LNHALCLRRG MFKSHCILNW PVQKERINAF HHKLQVALGS 900RNSAH 904 SEQ ID NO:23     Murine TLR3 nucleotidetagaatatga tacagggatt gcacccataa tctgggctga atcatgaaag ggtgttcctc 60ttatctaatg tactcctttg ggggactttt gtccctatgg attcttctgg tgtcttccac 120aaaccaatgc actgtgagat acaacgtagc tgactgcagc catttgaagc taacacacat 180acctgatgat cttccctcta acataacagt gttgaatctt actcacaacc aactcagaag 240attaccacct accaacttta caagatacag ccaacttgct atcttggatg caggatttaa 300ctccatttca aaactggagc cagaactgtg ccaaatactc cctttgttga aagtattgaa 360cctgcaacat aatgagctct ctcagatttc tgatcaaacc tttgtcttct gcacgaacct 420gacagaactc gatctaatgt ctaactcaat acacaaaatt aaaagcaacc ctttcaaaaa 480ccagaagaat ctaatcaaat tagatttgtc tcataatggt ttatcatcta caaagttggg 540aacgggggtc caactggaga acctccaaga actgctctta gcaaaaaata aaatccttgc 600gttgcgaagt gaagaacttg agtttcttgg caattcttct ttacgaaagt tggacttgtc 660atcaaatcca cttaaagagt tctccccggg gtgtttccag acaattggca agttattcgc 720cctcctcttg aacaacgccc aactgaaccc ccacctcaca gagaagcttt gctgggaact 780ttcaaacaca agcatccaga atctctctct ggctaacaac cagctgctgg ccaccagcga 840gagcactttc tctgggctga agtggacaaa tctcacccag ctcgatcttt cctacaacaa 900cctccatgat gtcggcaacg gttccttctc ctatctccca agcctgaggt atctgtctct 960ggagtacaac aatatacagc gtctgtcccc tcgctctttt tatggactct ccaacctgag 1020gtacctgagt ttgaagcgag catttactaa gcaaagtgtt tcacttgctt cacatcccaa 1080cattgacgat ttttcctttc aatggttaaa atatttggaa tatctcaaca tggatgacaa 1140taatattcca agtaccaaaa gcaatacctt cacgggattg gtgagtctga agtacctaag 1200tctttccaaa actttcacaa gtttgcaaac tttaacaaat gaaacatttg tgtcacttgc 1260tcattctccc ttgctcactc tcaacttaac gaaaaatcac atctcaaaaa tagcaaatgg 1320tactttctct tggttaggcc aactcaggat acttgatctc ggccttaatg aaattgaaca 1380aaaactcagc ggccaggaat ggagaggtct gagaaatata tttgagatct acctatccta 1440taacaaatac ctccaactgt ctaccagttc ctttgcattg gtccccagcc ttcaaagact 1500gatgctcagg agggtggccc ttaaaaatgt ggatatctcc ccttcacctt tccgccctct 1560tcgtaacttg accattctgg acttaagcaa caacaacata gccaacataa atgaggactt 1620gctggagggt cttgagaatc tagaaatcct ggattttcag cacaataact tagccaggct 1680ctggaaacgc gcaaaccccg gtggtcccgt taatttcctg aaggggctgt ctcacctcca 1740catcttgaat ttagagtcca acggcttaga tgaaatccca gtcggggttt tcaagaactt 1800attcgaacta aagagcatca atctaggact gaataactta aacaaacttg aaccattcat 1860ttttgatgac cagacatctc taaggtcact gaacctccag aagaacctca taacatctgt 1920tgagaaggat gttttcgggc cgccttttca aaacctgaac agtttagata tgcgcttcaa 1980tccgttcgac tgcacgtgtg aaagtatttc ctggtttgtt aactggatca accagaccca 2040cactaatatc tttgagctgt ccactcacta cctctgtaac actccacatc attattatgg 2100cttccccctg aagcttttcg atacatcatc ctgtaaagac agcgccccct ttgaactcct 2160cttcataatc agcaccagta tgctcctggt ttttatactt gtggtactgc tcattcacat 2220cgagggctgg aggatctctt tttactggaa tgtttcagtg catcggattc ttggtttcaa 2280ggaaatagac acacaggctg agcagtttga atatacagcc tacataattc atgcccataa 2340agacagagac tgggtctggg aacatttctc cccaatggaa gaacaagacc aatctctcaa 2400attttgccta gaagaaaggg actttgaagc aggcgtcctt ggacttgaag caattgttaa 2460tagcatcaaa agaagccgaa aaatcatttt cgttatcaca caccatttat taaaagaccc 2520tctgtgcaga agattcaagg tacatcacgc agttcagcaa gctattgagc aaaatctgga 2580ttcaattata ctgatttttc tccagaatat tccagattat aaactaaacc atgcactctg 2640tttgcgaaga ggaatgttta aatctcattg catcttgaac tggccagttc agaaagaacg 2700gataaatgcc tttcatcata aattgcaagt agcacttgga tctcggaatt cagcacatta 2760aactcatttg aagatttgga gtcggtaaag ggatagatcc aatttataaa ggtccatcat 2820gaatctaagt tttacttgaa agttttgtat atttatttat atgtatagat gatgatatta 2880catcacaatc caatctcagt tttgaaatat ttcggcttat ttcattgaca tctggtttat 2940tcactccaaa taaacacatg ggcagttaaa aacatcctct attaatagat tacccattaa 3000ttcttgaggt gtatcacagc tttaaagggt tttaaatatt tttatataaa taagactgag 3060agttttataa atgtaatttt ttaaaactcg agtcttactg tgtagctcag aaaggcctgg 3120aaattaatat attagagagt catgtcttga acttatttat ctctgcctcc ctctgtctcc 3180agagtgttgc ttttaagggc atgtagcacc.acacccagct atgtacgtgt gggattttat 3240aatgctcatt tttgagacgt ttatagaata aaagataatt gcttttatgg tataaggcta 3300cttgaggtaa 3310

Nucleotide and amino acid sequences of human and murine TLR7 are known.See, for example, GenBank Accession Nos. AF240467, AF245702,NM_(—)016562, AF334942, NM_(—)133211; and AAF60188, AAF78035,NP_(—)057646, AAL73191, AAL73192. Human TLR7 is reported to be 1049amino acids long and to have a sequence provided in SEQ ID NO:24. Acorresponding nucleotide sequence is provided as SEQ ID NO:25. MurineTLR7 is reported to be 1050 amino acids long and to have a sequence asprovided in SEQ ID NO:26. A corresponding nucleotide sequence isprovided as SEQ ID NO:27. TLR7 polypeptide includes an extracellulardomain having leucine-rich repeat region, a transmembrane domain, and anintracellular domain that includes a TIR domain.

As used herein a “TLR7 polypeptide” refers to a polypeptide including afull-length TLR7 according to one of the sequences above, orthologs,allelic variants, SNPs, variants incorporating conservative amino acidsubstitutions, TLR7 fusion proteins, and functional fragments of any ofthe foregoing. Preferred embodiments include human TLR7 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the human TLR7 amino acid sequence of SEQ IDNO:24. Preferred embodiments also include murine TLR7 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the murine TLR7 amino acid sequence of SEQ IDNO:26.

As used herein “TLR7 signaling” refers to an ability of a TLR7polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, alsoreferred to herein as the TLR signal transduction pathway. Changes inTLR7 activity can be measured by assays such as those disclosed herein,including expression of genes under control of κB-sensitive promotersand enhancers. Such naturally occurring genes include the genes encodingIL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), andthe costimulatory molecules CD80 and CD86. Other genes can be placedunder the control of such regulatory elements (see below) and thus serveto report the level of TLR7 signaling. Additional nucleotide sequencecan be added to SEQ ID NO:25 or SEQ ID NO:27, preferably to the 5′ orthe 3′ end of the open reading frame of SEQ ID NO:25, to yield anucleotide sequence encoding a chimeric polypeptide that includes adetectable or reporter moiety, e.g., FLAG, luciferase (luc), greenfluorescent protein (GFP), and others known by those skilled in the art.

SEQ ID NO:24     Human TLR7 amino acidMVFPMWTLKR QILILFNIIL ISKLLGARWF PKTLPCDVTL DVPKNHVIVD CTDKHLTEIP 60GGIPTNTTNL TLTINHIPDI SPASFHRLDH LVEIDFRCNC VPIPLGSKNN MCIKRLQIKP 120RSFSGLTYLK SLYLDGNQLL EIPQGLPPSL QLLSLEANNI FSIRKENLTE LANIEILYLG 180QNCYYRNPCY VSYSIEKDAF LNLTKLKVLS LKDNNVTAVP TVLPSTLTEL YLYNNMIAKI 240QEDDFNNLNQ LQILDLSGNC PRCYNAPFPC APCKNNSPLQ IPVNAFDALT ELKVLRLHSN 300SLQEVPPRWF KNINKLQELD LSQNFLAKEI GDAKFLHFLP SLIQLDLSFN FELQVYRASM 360NLSQAFSSLK SLKILRIRGY VFKELKSFNL SPLHNLQNLE VLDLGTNFIK IANLSMFKQF 420KRLKVIDLSV NKISPSGDSS EVGFCSNART SVESYEPQVL EQLHYFRYDK YARSCRFKNK 480EASFMSVNES CYKYGQTLDL SKNSIFFVKS SDFQHLSFLK CLNLSGNLIS QTLNGSEFQP 540LAELRYLDFS NNRLDLLHST AFEELHKLEV LDISSNSHYF QSEGITHMLN FTKNLKVLQK 600LMMNDNDISS STSRTMESES LRTLEFRGNH LDVLWREGDN RYLQLFKNLL KLEELDISKN 660SLSFLPSGVF DGMPPNLKNL SLAKNGLKSF SWKKLQCLKN LETLDLSHNQ LTTVPERLSN 720CSRSLKNLIL KNNQIRSLTK YFLQDAFQLR YLDLSSNKIQ MIQKTSFPEN VLNNLKMLLL 780HHNRFLCTCD AVWFVWWVNH TEVTIPYLAT DVTCVGPGAH KGQSVISLDL YTCELDLTNL 840ILFSLSISVS LFLMVMMTAS HLYFWDVWYI YHFCKAKIKG YQRLISPDCC YDAFIVYDTK 900DPAVTEWVLA ELVAKLEDPR EKHFNLCLEE RDWLPGQPVL ENLSQSIQLS KKTVFVMTDK 960YAKTENFKIA FYLSHQRLMD EKVDVIILIF LEKPFQKSKF LQLRKRLCGS SVLEWPTNPQ 1020AHPYFWQCLK NALATDNHVA YSQVFKETV 1049SEQ ID NO:25     Human TLR7 nucleotideactccagata taggatcact ccatgccatc aagaaagttg atgctattgg gcccatctca 60agctgatctt ggcacctctc atgctctgct ctcttcaacc agacctctac attccatttt 120ggaagaagac taaaaatggt gtttccaatg tggacactga agagacaaat tcttatcctt 180tttaacataa tcctaatttc caaactcctt ggggctagat ggtttcctaa aactctgccc 240tgtgatgtca ctctggatgt tccaaagaac catgtgatcg tggactgcac agacaagcat 300ttgacagaaa ttcctggagg tattcccacg aacaccacga acctcaccct caccattaac 360cacataccag acatctcccc agcgtccttt cacagactgg accatctggt agagatcgat 420ttcagatgca actgtgtacc tattccactg gggtcaaaaa acaacatgtg catcaagagg 480ctgcagatta aacccagaag ctttagtgga ctcacttatt taaaatccct ttacctggat 540ggaaaccagc tactagagat accgcagggc ctcccgccta gcttacagct tctcagcctt 600gaggccaaca acatcttttc catcagaaaa gagaatctaa cagaactggc caacatagaa 660atactctacc tgggccaaaa ctgttattat cgaaatcctt gttatgtttc atattcaata 720gagaaagatg ccttcctaaa cttgacaaag ttaaaagtgc tctccctgaa agataacaat 780gtcacagccg tccctactgt tttgccatct actttaacag aactatatct ctacaacaac 840atgattgcaa aaatccaaga agatgatttt aataacctca accaattaca aattcttgac 900ctaagtggaa attgccctcg ttgttataat gccccatttc cttgtgcgcc gtgtaaaaat 960aattctcccc tacagatccc tgtaaatgct tttgatgcgc tgacagaatt aaaagtttta 1020cgtctacaca gtaactctct tcagcatgtg cccccaagat ggtttaagaa catcaacaaa 1080ctccaggaac tggatctgtc ccaaaacttc ttggccaaag aaattgggga tgctaaattt 1140ctgcattttc tccccagcct catccaattg gatctgtctt tcaattttga acttcaggtc 1200tatcgtgcat ctatgaatct atcacaagca ttttcttcac tgaaaagcct gaaaattctg 1260cggatcagag gatatgtctt taaagagttg aaaagcttta acctctcgcc attacataat 1320cttcaaaatc ttgaagttct tgatcttggc actaacttta taaaaattgc taacctcagc 1380atgtttaaac aatttaaaag actgaaagtc atagatcttt cagtgaataa aatatcacct 1440tcaggagatt caagtgaagt tggcttctgc tcaaatgcca gaacttctgt agaaagttat 1500gaaccccagg tcctggaaca attacattat ttcagatatg ataagtatgc aaggagttgc 1560agattcaaaa acaaagaggc ttctttcatg tctgttaatg aaagctgcta caagtatggg 1620cagaccttgg atctaagtaa aaatagtata ttttttgtca agtcctctga ttttcagcat 1680ctttctttcc tcaaatgcct gaatctgtca ggaaatctca ttagccaaac tcttaatggc 1740agtgaattcc aacctttagc agagctgaga tatttggact tctccaacaa ccggcttgat 1800ttactccatt caacagcatt tgaagagctt cacaaactgg aagttctgga tataagcagt 1860aatagccatt attttcaatc agaaggaatt actcatatgc taaactttac caagaaccta 1920aaggttctgc agaaactgat gatgaacgac aatgacatct cttcctccac cagcaggacc 1980atggagagtg agtctcttag aactctggaa ttcagaggaa atcacttaga tgttttatgg 2040agagaaggtg ataacagata cttacaatta ttcaagaatc tgctaaaatt agaggaatta 2100gacatctcta aaaattccct aagtttcttg ccttctggag tttttgatgg tatgcctcca 2160aatctaaaga atctctcttt ggccaaaaat gggctcaaat ctttcagttg gaagaaactc 2220cagtgtctaa agaacctgga aactttggac ctcagccaca accaactgac cactgtccct 2280gagagattat ccaactgttc cagaagcctc aagaatctga ttcttaagaa taatcaaatc 2340aggagtctga cgaagtattt tctacaagat gccttccagt tgcgatatct ggatctcagc 2400tcaaataaaa tccagatgat ccaaaagacc agcttcccag aaaatgtcct caacaatctg 2460aagatgttgc ttttgcatca taatcggttt ctgtgcacct gtgatgctgt gtggtttgtc 2520tggtgggtta accatacgga ggtgactatt ccttacctgg ccacagatgt gacttgtgtg 2580gggccaggag cacacaaggg ccaaagtgtg atctccctgg atctgtacac ctgtgagtta 2640gatctgacta acctgattct gttctcactt tccatatctg tatctctctt tctcatggtg 2700atgatgacag caagtcacct ctatttctgg gatgtgtggt atatttacca tttctgtaag 2760gccaagataa aggggtatca gcgtctaata tcaccagact gttgctatga tgcttttatt 2820gtgtatgaca ctaaagaccc agctgtgacc gagtgggttt tggctgagct ggtggccaaa 2880ctggaagacc caagagagaa acattttaat ttatgtctcg aggaaaggga ctggttacca 2940gggcagccag ttctggaaaa cctttcccag agcatacagc ttagcaaaaa gacagtgttt 3000gtgatgacag acaagtatgc aaagactgaa aattttaaga tagcatttta cttgtcccat 3060cagaggctca tggatgaaaa agttgatgtg attatcttga tatttcttga gaagcccttt 3120cagaagtcca agttcctcca gctccggaaa aggctctgtg ggagttctgt ccttgagtgg 3180ccaacaaacc cgcaagctca cccatacttc tggcagtgtc taaagaacgc cctggccaca 3240gacaatcatg tggcctatag tcaggtgttc aaggaaacgg tctagccctt ctttgcaaaa 3300cacaactgcc tagtttacca aggagaggcc tggctgttta aattgttttc atatatatca 3360caccaaaagc gtgttttgaa attcttcaag aaatgagatt gcccatattt caggggagcc 3420accaacgtct gtcacaggag ttggaaagat ggggtttata taatgcatca agtcttcttt 3480cttatctctc tgtgtctcta tttgcacttg agtctctcac ctcagctcct gtaaaagagt 3540ggcaagtaaa aaacatgggg ctctgattct cctgtaattg tgataattaa atatacacac 3600aatcatgaca ttgagaagaa ctgcatttct acccttaaaa agtactggta tatacagaaa 3660tagggttaaa aaaaactcaa gctctctcta tatgagacca aaatgtacta gagttagttt 3720agtgaaataa aaaaccagtc agctggccgg gcatggtggc tcatgcttgt aatcccagca 3780ctttgggagg ccgaggcagg tggatcacga ggtcaggagt ttgagaccag tctggccaac 3840atggtgaaac cccgtctgta ctaaaaatac aaaaattagc tgggcgtggt ggtgggtgcc 3900tgtaatccca gctacttggg aggctgaggc aggagaatcg cttgaacccg ggaggtggag 3960gtggcagtga gccgagatca cgccactgca atgcagcccg ggcaacagag ctagactgtc 4020tcaaaagaac aaaaaaaaaa aaacacaaaa aaactcagtc agcttcttaa ccaattgctt 4080ccgtgtcatc cagggcccca ttctgtgcag attgagtgtg ggcaccacac aggtggttgc 4140tgcttcagtg cttcctgctc tttttccttg ggcctgcttc tgggttccat agggaaacag 4200taagaaagaa agacacatcc ttaccataaa tgcatatggt ccacctacaa atagaaaaat 4260atttaaatga tctgccttta tacaaagtga tattctctac ctttgataat ttacctgctt 4320aaatgttttt atctgcactg caaagtactg tatccaaagt aaaatttcct catccaatat 4380ctttcaaact gttttgttaa ctaatgccat atatttgtaa gtatctgcac acttgataca 4440gcaacgttag atggttttga tggtaaaccc taaaggagga ctccaagagt gtgtatttat 4500ttatagtttt atcagagatg acaattattt gaatgccaat tatatggatt cctttcattt 4560tttgctggag gatgggagaa gaaaccaaag tttatagacc ttcacattga gaaagcttca 4620gttttgaact tcagctatca gattcaaaaa caacagaaag aaccaagaca ttcttaagat 4680gcctgtactt tcagctgggt ataaattcat gagttcaaag attgaaacct gaccaatttg 4740ctttatttca tggaagaagt gatctacaaa ggtgtttgtg ccatttggaa aacagcgtgc 4800atgtgttcaa gccttagatt ggcgatgtcg tattttcctc acgtgtggca atgccaaagg 4860ctttacttta cctgtgagta cacactatat gaattatttc caacgtacat ttaatcaata 4920agggtcacaa attcccaaat caatctctgg aataaataga gaggtaatta aattgctgga 4980gccaactatt tcacaacttc tgtaagc 5007SEQ ID NO:26     Murine TLR7 amino acidMVFSMWTRKR QILIFLNMLL VSRVFGFRWF PKTLPCEVKV NIPEAHVIVD CTDKHLTEIP 60EGIPTNTTNL TLTINHIPSI SPDSFRRLNH LEEIDLRCNC VPVLLGSKAN VCTKRLQIRP 120GSFSGLSDLK ALYLDGNQLL EIPQDLPSSL HLLSLEANNI FSITKENLTE LVNIETLYLG 180QNCYYRNPCN VSYSIEKDAF LVMRNLKVLS LKDNNVTAVP TTLPPNLLEL YLYNNIIKKI 240QENDFNNLNE LQVLDLSGNC PRCYNVPYPC TPCENNSPLQ IHDNAFNSLT ELKVLRLHSN 300SLQHVPPTWF KNMRNLQELD LSQNYLAREI EEAKFLHFLP NLVELDFSFN YELQVYHASI 360TLPHSLSSLE NLKILRVKGY VFKELKNSSL SVLHKLPRLE VLDLGTNFIK IADLNIFKHF 420ENLKLIDLSV NKISPSEESR EVGFCPNAQT SVDRHGPQVL EALHYFRYDE YARSCRFKNK 480EPPSFLPLNA DCHIYGQTLD LSRNNIFFIK PSDFQHLSFL KCLNLSGNTI GQTLNGSELW 540PLRELRYLDF SNNRLDLLYS TAFEELQSLE VLDLSSNSHY FQAEGITHML NFTKKLRLLD 600KLMMNDNDIS TSASRTMESD SLRILEFRGN HLDVLWRAGD NRYLDFFKNL FNLEVLDISR 660NSLNSLPPEV FEGMPPNLKN LSLAKNGLKS FFWDRLQLLK HLEILDLSHN QLTKVPERLA 720NCSKSLTTLI LKHNQIRQLT KYFLEDALQL RYLDISSNKI QVIQKTSFPE NVLNNLEMLV 780LHHNRFLCNC DAVWFVWWVN ETDVTIPYLA TDVTCVGPGA HKGQSVISLD LYTCELDLTN 840LILFSVSISS VLFLMVVMTT SHLFFWDMWY IYYFWKAKIK GYQHLQSMES CYDAFIVYDT 900KNSAVTEWVL QELVAKLEDP REKHFNLCLE ERDWLPGQPV LENLSQSIQL SKKTVFVMTQ 960KYAKTESFKM AFYLSHQRLL DEKVDVIILI FLEKPLQKSK FLQLRKRLCR SSVLEWPANP 1020QAHPYFWQCL KNALTTDNHV AYSQMFKETV 1050SEQ ID NO:27     Murine TLR7 nucleotideattctcctcc accagacctc ttgattccat tttgaaagaa aactgaaaat ggtgttttcg 60atgtggacac ggaagagaca aattttgatc tttttaaata tgctcttagt ttctagagtc 120tttgggtttc gatggtttcc taaaactcta ccttgtgaag ttaaagtaaa tatcccagag 180gcccatgtga tcgtggactg cacagacaag catttgacag aaatccctga gggcattccc 240actaacacca ccaatcttac ccttaccatc aaccacatad caagcatctc tccagattcc 300ttccgtaggc tgaaccatct ggaagaaatc gatttaagat gcaattgtgt acctgttcta 360ctggggtcca aagccaatgt gtgtaccaag aggctgcaga ttagacctgg aagctttagt 420ggactctctg acttaaaagc cctttacctg gatggaaacc aacttctgga gataccacag 480gatctgccat ccagcttaca tcttctgagc cttgaggcta acaacatctt ctccatcacg 540aaggagaatc taacagaact ggtcaacatt gaaacactct acctgggtca aaactgttat 600tatcgaaatc cttgcaatgt ttcctattct attgaaaaag atgctttcct agttatgaga 660aatttgaagg ttctctcact aaaagataac aatgtcacag ctgtccccac cactttgcca 720cctaatttac tagagctcta tctttataac aatatcatta agaaaatcca agaaaatgat 780tttaataacc tcaatgagtt gcaagttctt gacctaagtg gaaattgccc tcgatgttat 840aatgtcccat atccgtgtac accgtgtgaa aataattccc ccttacagat ccatgacaat 900gctttcaatt cattgacaga attaaaagtt ttacgtttac acagtaattc tcttcagcat 960gtgcccccaa catggtttaa aaacatgaga aacctccagg aactagacct ctcccaaaac 1020tacttggcca gagaaattga ggaggccaaa tttttgcatt ttcttcccaa ccttgttgag 1080ttggattttt ctttcaatta tgagctgcag gtctaccatg catctataac tttaccacat 1140tcactctctt cattggaaaa cttgaaaatt ctgcgtgtca aggggtatgt ctttaaagag 1200ctgaaaaact ccagtctttc tgtattgcac aagcttccca ggctggaagt tcttgacctt 1260ggcactaact tcataaaaat tgctgacctc aacatattca aacattttga aaacctcaaa 1320ctcatagacc tttcagtgaa taagatatct ccttcagaag agtcaagaga agttggcttt 1380tgtcctaatg ctcaaacttc tgtagaccgt catgggcccc aggtccttga ggccttacac 1440tatttccgat acgatgaata tgcacggagc tgcaggttca aaaacaaaga gccaccttct 1500ttcttgcctt tgaatgcaga ctgccacata tatgggcaga ccttagactt aagtagaaat 1560aacatatttt ttattaaacc ttctgatttt cagcatcttt cattcctcaa atgcctcaac 1620ttatcaggaa acaccattgg ccaaactctt aatggcagtg aactctggcc gttgagagag 1680ttgcggtact tagacttctc caacaaccgg cttgatttac tctactcaac agcctttgaa 1740gagctccaga gtcttgaagt tctggatcta agtagtaaca gccactattt tcaagcagaa 1800ggaattactc acatgctaaa ctttaccaag aaattacggc ttctggacaa actcatgatg 1860aatgataatg acatctctac ttcggccagc aggaccatgg aaagtgactc tcttcgaatt 1920ctggagttca gaggcaacca tttagatgtt ctatggagag ccggtgataa cagatacttg 1980gacttcttca agaatttgtt caatttagag gtattagata tctccagaaa ttccctgaat 2040tccttgcctc ctgaggtttt tgagggtatg ccgccaaatc taaagaatct ctccttggcc 2100aaaaatgggc tcaaatcttt cttttgggac agactccagt tactgaagca tttggaaatt 2160ttggacctca gccataaCca gctgacaaaa gtacctgaga gattggccaa ctgttccaaa 2220agtctcacaa cactgattCt taagcataat caaatcaggc aattgacaaa atattttcta 2280gaagatgctt tgcaattgCg ctatctagac atcagttcaa ataaaatcca ggtcattcag 2340aagactagct tcccagaaaa tgtcctcaac aatctggaga tgttggtttt acatcacaat 2400cgctttcttt gcaactgtga tgctgtgtgg tttgtctggt gggttaacca tacagatgtt 2460actattccat acctggccac tgatgtgact tgtgtaggtc caggagcaca caaaggtcaa 2520agtgtcatat cccttgatct gtatacgtgt gagttagatc tcacaaacct gattctgttc 2580tcagtttcca tatcatcagt cctctttctt atggtagtta tgacaacaag tcacctcttt 2640ttctgggata tgtggtacat ttattatttt tggaaagcaa agataaaggg gtatcagcat 2700ctgcaatcca tggagtcttg ttatgatgct tttattgtgt atgacactaa aaactcagct 2760gtgacagaat gggttttgca ggagctggtg gcaaaattgg aagatccaag agaaaaacac 2820ttcaatttgt gtctagaaga aagagactgg ctaccaggac agccagttct agaaaacctt 2880tcccagagca tacagctcag caaaaagaca gtgtttgtga tgacacagaa atatgctaag 2940actgagagtt ttaagatggc attttatttg tctcatcaga ggctcctgga tgaaaaagtg 3000gatgtgatta tcttgatatt cttggaaaag cctcttcaga agtctaagtt tcttcagctc 3060aggaagagac tctgcaggag ctctgtcctt gagtggcctg caaatccaca ggctcaccca 3120tacttctggc agtgcctgaa aaatgccctg accacagaca atcatgtggc ttatagtcaa 3180atgttcaagg aaacagtcta gctctctgaa gaatgtcacc acctaggaca tgccttgaat 3240cga 3243

Nucleotide and amino acid sequences of human and murine TLR8 are known.See, for example, GenBank Accession Nos. AF246971, AF245703,NM_(—)016610, XM_(—)045706, AY035890, NM_(—)133212; and AAF64061,AAF78036, NP 057694, XP_(—)045706, AAK62677, NP_(—)573475. Human TLR8 isreported to exist in at least two isoforms, one 1041 amino acids longhaving a sequence provided in SEQ ID NO:28, and the other 1059 aminoacids long having a sequence as provided in SEQ ID NO:30. Correspondingnucleotide sequences are provided as SEQ ID NO:29 and SEQ ID NO:31,respectively. The shorter of these two isoforms is believed to be moreimportant. Murine TLR8 is 1032 amino acids long and has a sequence asprovided in SEQ ID NO:32. The corresponding nucleotide sequence isprovided as SEQ ID NO:33. TLR8 polypeptide includes an extracellulardomain having leucine-rich repeat region, a transmembrane domain, and anintracellular domain that includes a TIR domain.

As used herein a “TLR8 polypeptide” refers to a polypeptide including afull-length TLR8 according to one of the sequences above, orthologs,allelic variants, SNPs, variants incorporating conservative amino acidsubstitutions, TLR8 fusion proteins, and functional fragments of any ofthe foregoing. Preferred embodiments include human TLR8 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the human TLR8 amino acid sequence of SEQ IDNO:28. Preferred embodiments also include murine TLR8 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the murine TLR8 amino acid sequence of SEQ IDNO:32.

As used herein “TLR8 signaling” refers to an ability of a TLR8polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, alsoreferred to herein as the TLR signal transduction pathway. Changes inTLR8 activity can be measured by assays such as those disclosed herein,including expression of genes under control of κB-sensitive promotersand enhancers. Such naturally occurring genes include the genes encodingIL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), andthe costimulatory molecules CD80 and CD86. Other genes can be placedunder the control of such regulatory elements (see below) and thus serveto report the level of TLR8 signaling. Additional nucleotide sequencecan be added to SEQ ID NO:29 or SEQ ID NO:33, preferably to the 5′ orthe 3′ end of the open reading frame of SEQ ID NO:29, to yield anucleotide sequence encoding a chimeric polypeptide that includes adetectable or reporter moiety, e.g., FLAG, luciferase (luc), greenfluorescent protein (GFP), and others known by those skilled in the art.

SEQ ID NO:28     Human TLR8 amino acid (1041)MENMFLQSSM LTCIFLLISG SCELCAEENF SRSYPCDEKK QNDSVIAECS NRRLQEVPQT 60VGKYVTELDL SDNFITHITN ESFQGLQNLT KINLNHNPNV QHQNGNPGIQ SNGLNITDGA 120FLNLKNLREL LLEDNQLPQI PSGLPESLTE LSLIQNNIYN ITKEGISRLI NLKNLYLAWN 180CYFNKVCEKT NIEDGVFETL TNLELLSLSF NSLSHVPPKL PSSLRKLFLS NTQIKYISEE 240DFKGLINLTL LDLSGNCPRC FNAPFPCVPC DGGASINIDR FAFQNLTQLR YLNLSSTSLR 300KINAAWFKNM PHLKVLDLEF NYLVGEIASG AFLTMLPRLE ILDLSFNYIK GSYPQHINIS 360RNFSKLLSLR ALHLRGYVFQ ELREDDFQPL MQLPNLSTIN LGINFIKQID FKLFQNFSNL 420EIIYLSENRI SPLVKDTRQS YANSSSFQRH IRKRRSTDFE FDPHSNFYHF TRPLIKPQCA 480AYGKALDLSL NSIFFIGPNQ FENLPDIACL NLSANSNAQV LSGTEFSAIP HVKYLDLTNN 540PLDFDNASAL TELSDLEVLD LSYNSHYFRI AGVTHHLEFI QNFTNLKVLN LSHNNIYTLT 600DKYNLESKSL VELVFSGNRL DILWNDDDNR YISIFKGLKN LTRLDLSLNR LKHIPNEAFL 660NLPASLTELH INDNMLKFFN WTLLQQFPRL ELLDLRGNKL LFLTDSLSDF TSSLRTLLLS 720HNRISHLPSG FLSEVSSLKH LDLSSNLLKT INKSALETKT TTKLSMLELH GNPFECTCDI 780GDFRRWMDEH LNVKIPRLVD VICASPGDQR GKSIVSLELT TCVSDVTAVI LFFFTFFITT 840MVMLAALAHH LFYWDVWFIY NVCLAKVKGY RSLSTSQTFY DAYISYDTKD ASVTDWVINE 900LRYHLEESRD KNVLLCLEER DWDPGLAIID NLMQSINQSK KTVFVLTKKY AKSWNFKTAF 960YLALQRLMDE NNDVIIFILL EPVLQHSQYL RLRQRICKSS ILQWPDNPKA EGLFWQTLRN 1020VVLTENDSRY NNMYVDSIKQ Y 1041 SEQ ID NO:29     Human TLR8 nucleotidettctgcgctg ctgcaagtta cggaatgaaa aattagaaca acagaaacat ggaaaacatg 60ttccttcagt cgtcaatgct gacctgcatt ttcctgctaa tatctggttc ctgtgagtta 120tgcgccgaag aaaatttttc tagaagctat ccttgtgatg agaaaaagca aaatgactca 180gttattgcag agtgcagcaa tcgtcgacta caggaagttc cccaaacggt gggcaaatat 240gtgacagaac tagacctgtc tgataatttc atcacacaca taacgaatga atcatttcaa 300gggctgcaaa atctcactaa aataaatcta aaccacaacc ccaatgtaca gcaccagaac 360ggaaatcccg gtatacaatc aaatggcttg aatatcacag acggggcatt cctcaaccta 420aaaaacctaa gggagttact gcttgaagac aaccagttac cccaaatacc ctctggtttg 480ccagagtctt tgacagaact tagtctaatt caaaacaata tatacaacat aactaaagag 540ggcatttcaa gacttataaa cttgaaaaat ctctatttgg cctggaactg ctatttt4ac 600aaagtttgcg agaaaactaa catagaagat ggagtatttg aaacgctgac aaatttggag 660ttgctatcac tatctttcaa ttctctttca cacgtgccac ccaaactgcc aagctcccta 720cgcaaacttt ttctgagcaa cacccagatc aaatacatta gtgaagaaga tttcaaggga 780ttgataaatt taacattact agatttaagc gggaactgtc cgaggtgctt caatgcccca 840tttccatgcg tgccttgtga tggtggtgct tcaattaata tagatcgttt tgcttttcaa 900aacttgaccc aacttcgata cctaaacctc tctagcactt ccctcaggaa gattaatgct 960gcctggttta aaaatatgcc tcatctgaag gtgctggatc ttgaattcaa ctatttagtg 1020ggagaaatag cctctggggc atttttaacg atgctgcccc gcttagaaat acttgacttg 1080tcttttaact atataaaggg gagttatcca cagcatatta atatttccag aaacttctct 1140aaacttttgt ctctacgggc attgcattta agaggttatg tgttccagga actcagagaa 1200gatgatttcc agcccctgat gcagcttcca aacttatcga ctatcaactt gggtattaat 1260tttattaagc aaatcgattt caaacttttc caaaatttct ccaatctgga aattatttac 1320ttgtcagaaa acagaatatc accgttggta aaagataccc ggcagagtta tgcaaatagt 1380tcctcttttc aacgtcatat ccggaaacga cgctcaacag attttgagtt tgacccacat 1440tcgaactttt atcatttcac ccgtccttta ataaagccac aatgtgctgc ttatggaaaa 1500gccttagatt taagcctcaa cagtattttc ttcattgggc caaaccaatt tgaaaatctt 1560cctgacattg cctgtttaaa tctgtctgca aatagcaatg ctcaagtgtt aagtggaact 1620gaattttcag ccattcctca tgtcaaatat ttggatttga caaacaatag actagacttt 1680gataatgcta gtgctcttac tgaattgtcc gacttggaag ttctagatct cagctataat 1740tcacactatt tcagaatagc aggcgtaaca catcatctag aatttattca aaatttcaca 1800aatctaaaag ttttaaactt gagccacaac aacatttata ctttaacaga taagtataac 1860ctggaaagca agtccctggt agaattagtt ttcagtggca atcgccttga cattttgtgg 1920aatgatgatg acaacaggta tatctccatt ttcaaaggtc tcaagaatct gacacgtctg 1980gatttatccc ttaataggct gaagcacatc ccaaatgaag cattccttaa tttgccagcg 2040agtctcactg aactacatat aaatgataat atgttaaagt tttttaactg gacattactc 2100cagcagttcc ctcgtctcga gttgcttgac ttacgtggaa acaaactact ctttttaact 2160gatagcctat ctgactttac atcttccctt cggacactgc tgctgagtca taacaggatt 2220tcccacctac cctctggctt tctttctgaa gtcagtagtc tgaagcacct cgatttaagt 2280tccaatctgc taaaaacaat caacaaatcc gcacttgaaa ctaagaccac caccaaatta 2340tctatgttgg aactacacgg aaaccccttt gaatgcacct gtgacattgg agatttccga 2400agatggatgg atgaacatct gaatgtcaaa attcccagac tggtagatgt catttgtgcc 2460agtcctgggg atcaaagagg gaagagtatt gtgagtctgg agctgacaac ttgtgtttca 2520gatgtcactg cagtgatatt atttttcttc acgttcttta tcaccaccat ggttatgttg 2580gctgccctgg ctcaccattt gttttactgg gatgtttggt ttatatataa tgtgtgttta 2640gctaaggtaa aaggctacag gtctctttcc acatcccaaa ctttctatga tgcttacatt 2700tcttatgaca ccaaagatgc ctctgttact gactgggtga taaatgagct gcgctaccac 2760cttgaagaga gccgagacaa aaacgttctc ctttgtctag aggagaggga ttgggacccg 2820ggattggcca tcatcgacaa cctcatgcag agcatcaacc aaagcaagaa aacagtattt 2880gttttaacca aaaaatatgc aaaaagctgg aactttaaaa cagcttttta cttggctttg 2940cagaggctaa tggatgagaa catggatgtg attatattta tcctgctgga gccagtgtta 3000cagcattctc agtatttgag gctacggcag cggatctgta agagctccat cctccagtgg 3060cctgacaacc cgaaggcaga aggcttgttt tggcaaactc tgagaaatgt ggtcttgact 3120gaaaatgatt cacggtataa caatatgtat gtcgattcca ttaagcaata ctaactgacg 3180ttaagtcatg atttcgcgcc ataataaaga tgcaaaggaa tgacatttct gtattagtta 3240tctattgcta tgtaacaaat tatcccaaaa cttagtggtt taaaacaaca catttgctgg 3300cccacagttt t 3311 SEQ ID NO:30     Human TLR8 amino acid (1059)MKESSLQNSS CSLGKETKKE NNFLQSSMLT CIFLLISGSC ELCAEENFSR SYPCDEKKQN 60DSVIAECSNR RLQEVPQTVG KYVTELDLSD NFITHITNES FQGLQNLTKI NLNHNPNVQH 120QNGNPGIQSN GLNITDGAFL NLKNLRELLL EDNQLPQIPS GLPESLTELS LIQNNIYNIT 180KEGISRLINL KNLYLAWNCY FNKVCEKTNI EDGVFETLTN LELLSLSFNS LSHVSPKLPS 240SLRKLFLSNT QIKYISEEDF KGLThLTLLD LSGNCPRCFN APFPCVPCDG GASINIDRFA 300FQNLTQLRYL NLSSTSLRKI NAAWFKNMPH LKVLDLEFNY LVGEIASGAF LTMLPRLEIL 360DLSFNYIKGS YPQHINISRN FSKPLSLRAL HLRGYVFQEL REDDFQPLMQ LPNLSTINLG 420INFIKQIDFK LFQNFSNLEI IYLSENRISP LVKDTRQSYA NSSSFQRHIR KRRSTDFEFD 480PHSNFYHFTR PLIKPQCAAY GKALDLSLNS IFFIGPNQFE NLPDIACLNL SANSNAQVLS 540GTEFSAIPHV KYLDLTNNRL DFDNASALTE LSDLEVLDLS YNSHYFRIAG VTHHLEFIQN 600FTNLKVLNLS RNNIYTLTDK YNLESKSLVE LVFSGNRLDI LWNDDDNRYI SIFKGLKNLT 660RLDLSLNRLK HIPNEAFLNL PASLTELHIN DNNLKFFNWT LLQQFPRLEL LDLRGNKLLF 720LTDSLSDFTS SLRTLLLSHN RISHLPSGFL SEVSSLKHLD LSSNLLKTIN KSALETKTTT 780KLSMLELHGN PFECTCDIGD FRRWNDEHLN VKIPRLVDVI CASPGDQRGK SIVSLELTTC 840VSDVTAVILF FFTFFITTMV MLAALAHHLF YWDVWFIYNV CLAKIKGYRS LSTSQTFYDA 900YISYDTKDAS VTDWVINELR YHLEESRDKN VLLCLEERDW DPGLAIIDNL MQSINQSKKT 960VFVLTKKYAK SWNFKTAFYL ALQRLMDENM DVIIFILLEP VLQHSQYLRL RQRICKSSIL 1020QWPDNPKAEG LFWQTLRNVV LTENDSRYNN MYVDSIKQY 1059SEQ ID NO:31     Human TLR8 nucleotidectcctgcata gagggtacca ttctgcgctg ctgcaagtta cggaatgaaa aattagaaca 60acagaaacgt ggttctcttg acacttcagt gttagggaac atcagcaaga cccatcccag 120gagaccttga aggaagcctt tgaaagggag aatgaaggag tcatctttgc aaaatagctc 180ctgcagcctg ggaaaggaga ctaaaaagga aaacatgttc cttcagtcgt caatgctgac 240ctgcattttc ctgctaatat ctggttcctg tgagttatgc gccgaagaaa atttttctag 300aagctatcct tgtgatgaga aaaagcaaaa tgactcagtt attgcagagt gcagcaatcg 360tcgactacag gaagttcccc aaacggtggg caaatatgtg acagaactag acctgtctga 420taatttcatc acacacataa cgaatgaatc atttcaaggg ctgcaaaatc tcactaaaat 480aaatctaaac cacaacccca atgtacagca ccagaacgga aatcccggta tacaatcaaa 540tggcttgaat atcacagacg gggcattcct caacctaaaa aacctaaggg agttactgct 600tgaagacaac cagttacccc aaataccctc tggtttgcca gagtctttga cagaacttag 660tctaattcaa aacaatatat acaacataac taaagagggc atttcaagac ttataaactt 720gaaaaatctc tatttggcct ggaactgcta ttttaacaaa gtttgcgaga aaactaacat 780agaagatgga gtatttgaaa cgctgacaaa tttggagttg ctatcactat ctttcaattc 840tctttcacac gtgtcaccca aactgccaag ctccctacgc aaactttttc tgagcaacac 900ccagatcaaa tacattagtg aagaagattt caagggattg ataaatttaa cattactaga 960tttaagcggg aactgtccga ggtgcttcaa tgccccattt ccatgcgtgc cttgtgatgg 1020tggtgcttca attaatatag atcgttttgc ttttcaaaac ttgacccaac ttcgatacct 1080aaacctctct agcacttccc tcaggaagat taatgctgcc tggtttaaaa atatgcctca 1140tctgaaggtg ctggatcttg aattcaacta tttagtggga gaaatagcct ctggggcatt 1200tttaacgatg ctgccccgct tagaaatact tgacttgtct tttaactata taaaggggag 1260ttatccacag catattaata tttccagaaa cttctctaaa cctttgtctc tacgggcatt 1320gcatttaaga ggttatgtgt tccaggaact cagagaagat gatttccagc ccctgatgca 1380gcttccaaac ttatcgacta tcaacttggg tattaatttt attaagcaaa tcgatttcaa 1440acttttccaa aatttctcca atctggaaat tatttacttg tcagaaaaca gaatatcacc 1500gttggtaaaa gatacccggc agagttatgc aaatagttcc tcttttcaac gtcatatccg 1560gaaacgacgc tcaacagatt ttgagtttga cccacattcg aacttttatc atttcacccg 1620tcctttaata aagccacaat gtgctgctta tggaaaagcc ttagatttaa gcctcaacag 1680tattttcttc attgggccaa accaatttga aaatcttcct gacattgcct gtttaaatct 1740gtctgcaaat agcaatgctc aagtgttaag tggaactgaa ttttcagcca ttcctcatgt 1800caaatatttg gatttgacaa acaatagact agactttgat aatgctagtg ctcttactga 1860attgtccgac ttggaagttc tagatctcag ctataattca cactatttca gaatagcagg 1920cgtaacacat catctagaat ttattcaaaa tttcacaaat ctaaaagttt taaacttgag 1980ccacaacaac atttatactt taacagataa gtataacctg gaaagcaagt ccctggtaga 2040attagttttc agtggcaatc gccttgacat tttgtggaat gatgatgaca acaggtatat 2100ctccattttc aaaggtctca agaatctgac acgtctggat ttatccctta ataggctgaa 2160gcacatccca aatgaagcat tccttaattt gccagcgagt ctcactgaac tacatataaa 2220tgataatatg ttaaagtttt ttaactggac attactccag cagtttcctc gtctcgagtt 2280gcttgactta cgtggaaaca aactactctt tttaactgat agcctatctg actttacatc 2340ttcccttcgg acactgctgc tgagtcataa caggatttcc cacctaccct ctggctttct 2400ttctgaagtc agtagtctga agcacctcga tttaagttcc aatctgctaa aaacaatcaa 2460caaatccgca cttgaaacta agaccaccac caaattatct atgttggaac tacacggaaa 2520cccctttgaa tgcacctgtg acattggaga tttccgaaga tggatggatg aacatctgaa 2580tgtcaaaatt cccagactgg tagatgtcat ttgtgccagt cctggggatc aaagagggaa 2640gagtattgtg agtctggagc taacaacttg tgtttcagat gtcactgcag tgatattatt 2700tttcttcacg ttctttatca ccaccatggt tatgttggct gccctggctc accatttgtt 2760ttactgggat gtttggttta tatataatgt gtgtttagct aagataaaag gctacaggtc 2820tctttccaca tcccaaactt tctatgatgc ttacatttct tatgacacca aagatgcctc 2880tgttactgac tgggtgataa atgagctgcg ctaccacctt gaagagagcc gagacaaaaa 2940cgttctcctt tgtctagagg agagggattg ggacccggga ttggccatca tcgacaacct 3000catgcagagc atcaaccaaa gcaagaaaac agtatttgtt ttaaccaaaa aatatgcaaa 3060aagctggaac tttaaaacag ctttttactt ggctttgcag aggctaatgg atgagaacat 3120ggatgtgatt atatttatcc tgctggagcc agtgttacag cattctcagt atttgaggct 3180acggcagcgg atctgtaaga gctccatcct ccagtggcct gacaacccga aggcagaagg 3240cttgttttgg caaactctga gaaatgtggt cttgactgaa aatgattcac ggtataacaa 3300tatgtatgtc gattccatta agcaatacta actgacgtta agtcatgatt tcgcgccata 3360ataaaga 3367 SEQ ID NO:32     Murine TLR8 amino acidMENMPPQSWO LTCFCLLSSG TSAIFHKANY SRSYPCDEIR HNSLVIAECN HRQLHEVPQT 60IGKYVTNIDL SDNAITHITK ESFZKLQNLT KIDLNHNAKQ QHPNENKNGM NITEGALLSL 120RNLTVLLLED NQLYTIPAGL PESLKELSLI QNNIFQVTKN NTFGLRNLER LYLGWNCYFK 180CNQTFKVEDG AFKNLIHLKV LSLSFNNLFY VPPKLPSSLR KLFLSNAKIM NITQEDFKGL 240ENLTLLDLSG NCPRCYNAPF PCTPCKENSS INIHPLAFQS LTQLLYLNLS STSLRTIPST 300WFENLSNLKE LELEFNYLVQ EIASGAFLTK LPSLQILDLS FNFQYKEYLQ FINISSNFSK 360LRSLKKLHLR GYVFRELKKK HFIFLQSLPN LATINLGINF IEKIDFKAFQ NFSKLDVIYL 420SGNRIASVLD GTDYSSWRNR LRKPLSTDDD EFDPHVNFYH STKPLIKPQC TAYGKALDLS 480LNNIFIIGKS QFEGFQDIAC LNLSFNANTQ VFNGTEFSSM PHIKYLDLTN NRLDFDDNNA 540FSDLHDLEVL DLSHNAHYFS IAGVTHRLGF IQNLINLRVL NLSHNGIYTL TEESELKSIS 600LKELVFSGNR LDHLWNANDG KYWSIFKSLQ NLIRLDLSYN NLQQIPNGAF LNLPQSLQEL 660LISGNKLRFF NWTLLQYFPH LHLLDLSRNE LYFLPNCLSK FAHSLETLLL SHNHFSHLPS 720GFLSEARNLV HLDLSFNTIK MINKSSLQTK MKTNLSILEL HGNYFDCTCD ISDFRSWLDE 780NLNITIPKLV NVICSNPGDQ KSKSIMSLDL TTCVSDTTAA VLFFLTFLTT SMVMLAALVH 840HLFYWDVWFI YHMCSAKLKG YRTSSTSQTF YDAYISYDTK DASVTDWVIN ELRYHLEESE 900DKSVLLCLEE RDWDPGLPII DNLMQSINQS KKTIFVLTKK YAKSWNFKTA FYLALQRLMD 960ENMDVIIFIL LEPVLQYSQY LRLRQRICKS SILQWPNNPK AENLFWQSLK NVVLTENDSR 1020YDDLYIDSIR QY 1032 SEQ ID NO:33     Murine TLR8 nucleotideattcagagtt ggatgttaag agagaaacaa acgttttacc ttcctttgtc tatagaacat 60ggaaaacatg ccccctcagt catggattct gacgtgcttt tgtctgctgt cctctggaac 120cagtgccatc ttccataaag cgaactattc cagaagctat ccttgtgacg agataaggca 180caactccctt gtgattgcag aatgcaacca tcgtcaactg catgaagttc cccaaactat 240aggcaagtat gtgacaaaca tagacttgtc agacaatgcc attacacata taacgaaaga 300gtcctttcaa aagctgcaaa acctcactaa aatcgatctg aaccacaatg ccaaacaaca 360gcacccaaat gaaaataaaa atggtatgaa tattacagaa ggggcacttc tcagcctaag 420aaatctaaca gttttactgc tggaagacaa ccagttatat actatacctg ctgggttgcc 480tgagtctttg aaagaactta gcctaattca aaacaatata tttcaggtaa ctaaaaacaa 540cacttttggg cttaggaact tggaaagact ctatttgggc tggaactgct attttaaatg 600taatcaaacc tttaaggtag aagatggggc atttaaaaat cttatacact tgaaggtact 660ctcattatct ttcaataacc ttttctatgt gccccccaaa ctaccaagtt ctctaaggaa 720actttttctg agtaatgcca aaatcatgaa catcactcag gaagacttca aaggactgga 780aaatttaaca ttactagatc tgagtggaaa ctgtccaagg tgttacaatg ctccatttcc 840ttgcacacct tgcaaggaaa actcatccat ccacatacat cctctggctt ttcaaagtct 900cacccaactt ctctatctaa acctttccag cacttccctc aggacgattc cttctacctg 960gtttgaaaat ctgtcaaatc tgaaggaact ccatcttgaa ttcaactatt tagttcaaga 1020aattgcctcg ggggcatttt taacaaaact acccagttta caaatccttg atttgtcctt 1080caactttcaa tataaggaat atttacaatt tattaatatt tcctcaaatt tctctaagct 1140tcgttctctc aagaagttgc acttaagagg ctatgtgttc cgagaactta aaaagaagca 1200tttcgagcat ctccagagtc ttccaaactt ggcaaccatc aacttgggca ttaactttat 1260tgagaaaatt gatttcaaag ctttccagaa tttttccaaa ctcgacgtta tctatttatc 1320aggaaatcgc atagcatctg tattagatgg tacagattat tcctcttggc gaaatcgtct 1380tcggaaacct ctctcaacag acgatgatga gtttgatcca cacgtgaatt tttaccatag 1440caccaaacct ttaataaagc cacagtgtac tgcttatggc aaggccttgg atttaagttt 1500gaacaatatt ttcattattg ggaaaagcca atttgaaggt tttcaggata tcgcctgctt 1560aaatctgtcc ttcaatgcca atactcaagt gtttaatggc acagaattct cctccatgcc 1620ccacattaaa tatttggatt taaccaacaa cagactagac tttgatgata acaatgcttt 1680cagtgatctt cacgatctag aagtgctgga cctgagccac aatgcacact atttcagtat 1740agcaggggta acgcaccgtc taggatttat ccagaactta ataaacctca gggtgttaaa 1800cctgagccac aatggcattt acaccctcac agaggaaagt gagctgaaaa gcatctcact 1860gaaagaattg gttttcagtg gaaatcgtct tgaccatttg tggaatgcaa atgatggcaa 1920atactggtcc atttttaaaa gtctccagaa tttgatacgc ctggacttat catacaataa 1980ccttcaacaa atcccaaatg gagcattcct caatttgcct cagagcctcc aagagttact 2040tatcagtggt aacaaattac gtttctttaa ttggacatta ctccagtatt ttcctcacct 2100tcacttgctg gatttatcga gaaatgagct gtattttcta cccaattgcc tatctaagtt 2160tgcacattcc ctggagacac tgctactgag ccataatcat ttctctcacc taccctctgg 2220cttcctctcc gaagccagga atctggtgca cctggatcta agtttcaaca caataaagat 2280gatcaataaa tcctccctgc aaaccaagat gaaaacgaac ttgtctattc tggagctaca 2340tgggaactat tttgactgca cgtgtgacat aagtgatttt cgaagctggc tagatgaaaa 2400tctgaatatc acaattccta aattggtaaa tgttatatgt tccaatcctg gggatcaaaa 2460atcaaagagt atcatgagcc tagatctcac gacttgtgta tcggatacca ctgcagctgt 2520cctgtttttc ctcacattcc ttaccacctc catggttatg ttggctgctc tggttcacca 2580cctgttttac tgggatgttt ggtttatcta tcacatgtgc tctgctaagt taaaaggcta 2640caggacttca tccacatccc aaactttcta tgatgcttat atttcttatg acaccaaaga 2700tgcatctgtt actgactggg taatcaatga actgcgctac caccttgaag agagtgaaga 2760caaaagtgtc ctcctttgtt tagaggagag ggattgggat ccaggattac ccatcattga 2820taacctcatg cagagcataa accagagcaa gaaaacaatc tttgttttaa ccaagaaata 2880tgccaagagc tggaacttta aaacagcttt ctacttggcc ttgcagaggc taatggatga 2940gaacatggat gtgattattt tcatcctcct ggaaccagtg ttacagtact cacagtacct 3000gaggcttcgg cagaggatct gtaagagctc catcctccag tggcccaaca atcccaaagc 3060agaaaacttg ttttggcaaa gtctgaaaaa tgtggtcttg actgaaaatg attcacggta 3120tgacgatttg tacattgatt ccattaggca atactagtga tgggaagtca cgactctgcc 3180atcataaaaa cacacagctt ctccttacaa tgaaccgaat 3220

Nucleotide and amino acid sequences of human and murine TLR9 are known.See, for example, GenBank Accession Nos. NM_(—)017442, AF259262,AB045180, AF245704, AB045181, AF348140, AF314224, NM_(—)031178; andNP_(—)059138, AAF 72189, BAB19259, AAF78037, BAB19260, AAK29625,AAK28488, NP_(—)112455. Human TLR9 is reported to exist in at least twoisoforms, one 1032 amino acids long having a sequence provided in SEQ IDNO:34, and the other 1055 amino acids long having a sequence as providedin SEQ ID NO:36. Corresponding nucleotide sequences are provided as SEQID NO:35 and SEQ ID NO:37, respectively. The shorter of these twoisoforms is believed to be more important. Murine TLR9 is 1032 aminoacids long and has a sequence as provided in SEQ ID NO:38. Acorresponding nucleotide sequence is provided as SEQ ID NO:39. TLR9polypeptide includes an extracellular domain having leucine-rich repeatregion, a transmembrane domain, and an intracellular domain thatincludes a TIR domain.

As used herein a “TLR9 polypeptide” refers to a polypeptide including afull-length TLR9 according to one of the sequences above, orthologs,allelic variants, SNPs, variants incorporating conservative amino acidsubstitutions, TLR9 fusion proteins, and functional fragments of any ofthe foregoing. Preferred embodiments include human TLR9 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the human TLR9 amino acid sequence of SEQ IDNO:34. Preferred embodiments also include murine TLR9 polypeptideshaving at least 65 percent sequence identity, more preferably at least80 percent sequence identity, even more preferably with at least 90percent sequence identity, and most preferably with at least 95 percentsequence identity with the murine TLR9 amino acid sequence of SEQ IDNO:38.

As used herein “TLR9 signaling” refers to an ability of a TLR9polypeptide to activate the TLR/IL-1R (TIR) signaling pathway, alsoreferred to herein as the TLR signal transduction pathway. Withoutmeaning to be held to any particular theory, it is believed that theTLR/IL-1R signaling pathway involves signaling via the molecules myeloiddifferentiation marker 88 (MyD88) and tumor necrosis factor (TNF)receptor-associated factor 6 (TRAF6), leading to activation of kinasesof the IκB kinase complex and the c-jun NH₂-terminal kinases (e.g., Jnk1/2). Häcker H et al. (2000) J Exp Med 192:595-600. Changes in TLR9activity can be measured by assays such as those disclosed herein,including expression of genes under control of κB-sensitive promotersand enhancers. Such naturally occurring genes include the genes encodingIL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12 p40), andthe costimulatory molecules CD80 and CD86. Other genes can be placedunder the control of such regulatory elements (see below) and thus serveto report the level of TLR9 signaling. Additional nucleotide sequencecan be added to SEQ ID NO:35 or SEQ ID NO:39, preferably to the 5′ orthe 3′ end of the open reading frame of SEQ ID NO:35, to yield anucleotide sequence encoding a chimeric polypeptide that includes adetectable or reporter moiety, e.g., FLAG, luciferase (luc), greenfluorescent protein (GFP), and others known by those skilled in the art.

SEQ ID NO:34     Human TLR9 amino acid (1032)MGFCRSALHP LSLLVQAIML AMTLALGTLP AFLPCELQPH GLVNCNWLFL KSVPHFSMAA 60PRGNVTSLSL SSNRIHHLHD SDFAKLPSLR HLNLKWNCPP VGLSPMHFPC HMTIEPSTFL 120AVPTLEELNL SYNNIMTVPA LPKSLISLSL SHTNILMLDS ASLAGLHALR FLFMDGNCYY 180KNPCRQALEV APGALLGLGN LTHLSLKYNN LTVVPRNLPS SLEYLLLSYN RIVKLAPEDL 240ANLTALRVLD VGGNCRRCDH APNPCMECPR HFPQLHPDTF SHLSRLEGLV LKDSSLSWLN 300ASWFRGLGNL RVLDLSENFL YKCITKTKAF QGLTQLRKLN LSFNYQKRVS FAHLSLAPSF 360GSLVALKELD MHGIFFRSLD ETTLRPLARL PMLQTLRLQM NFINQAQLGI FRAFPGLRYV 420DLSDNRISGA SELTATMGEA DGGEKVWLQP GDLAPAPVDT PSSEDFRPNC STLNFTLDLS 480RNNLVTVQPE MFAQLSHLQC LRLSHNCISQ AVNGSQFLPL TGLQVLDLSH NKLDLYHEHS 540FTELPRLEAL DLSYNSQPFG MQGVGHWFSF VAHLRTLRHL SLAHNNIHSQ VSQQLCSTSL 600RALDFSGNAL GHMWAEGDLY LHFFQGLSGL IWLDLSQNRL HTLLPQTLRN LPKSLQVLRL 660RDNYLAFFKW WSLHFLPKLE VLDLAGNQLK ALTNGSLPAG TRLRRLDVSC NSISFVAPGF 720FSKAKELREL NLSANALKTV DHSWFGPLAS ALQILDVSAN PLHCACGAAF MDFLLEVQAA 780VPGLPSRVKC GSPGQLQGLS IFAQDLRLCL DEALSWDCFA LSLLAVALGL GVPMLHHLCG 840WDLWYCFHLC LAWLPWRGRQ SGRDEDALPY DAFVVFDKTQ SAVADWVYNE LRGQLEECRG 900RWALRLCLEE RDWLPGKTLF ENLWASVYGS RKTLFVLAHT DRVSGLLRAS FLLAQQRLLE 960DRKDVVVLVI SLPDGRRSRY VRLRQRLCRQ SVLLWPHQPS GQRSFWAQLG MALTRDNHHF 1020YNRNFCQGPT AE 1032 SEQ ID NO:35     Human TLR9 nucleotideccgctgctgc ccctgtggga agggacctcg agtgtgaagc atccttccct gtagctgctg 60tccagtctgc ccgccagacc ctctggagaa gcccctgccc cccagcatgg gtttctgccg 120cagcgccctg cacccgctgt ctctcctggt gcaggccatc atgctggcca tgaccctggc 180cctgggtacc ttgcctgcct tcctaccctg tgagctccag ccccacggcc tggtgaactg 240caactggctg ttcctgaagt ctgtgcccca cttctccatg gcagcacccc gtggcaatgt 300caccagcctt tccttgtcct ccaaccgcat ccaccacctc catgattctg actttgccca 360cctgcccagc ctgcggcatc tcaacctcaa gtggaactgc ccgccggttg gcctcagccc 420catgcacttc ccctgccaca tgaccatcga gcccagcacc ttcttggctg tgcccaccct 480ggaagagcta aacctgagct acaacaacat catgactgtg cctgcgctgc ccaaatccct 540catatccctg tccctcagcc ataccaacat cctgatgcta gactctgcca gcctcgccgg 600cctgcatgcc ctgcgcttcc tattcatgga cggcaactgt tattacaaga acccctgcag 660gcaggcactg gaggtggccc cgggtgccct ccttggcctg ggcaacctca cccacctgtc 720actcaagtac aacaacctca ctgtggtgcc ccgcaacctg ccttccagcc tggagtatct 780gctgttgtcc tacaaccgca tcgtcaaact ggcgcctgag gacctggcca atctgaccgc 840cctgcgtgtg ctcgatgtgg gcggaaattg ccgccgctgc gaccacgctc ccaacccctg 900catggagtgc cctcgtcact tcccccagct acatcccgat accttcagcc acctgagccg 960tcttgaaggc ctggtgttga aggacagttc tctctcctgg ctgaatgcca gttggttccg 1020tgggctggga aacctccgag tgctggacct gagtgagaac ttcctctaca aatgcatcac 1080taaaaccaag gccttccagg gcctaacaca gctgcgcaag cttaacctgt ccttcaatta 1140ccaaaagagg gtgtcctttg cccacctgtc tctggcccct tccttcggga gcctggtcgc 1200cctgaaggag ctggacatgc acggcatctt cttccgctca ctcgatgaga ccacgctccg 1260gccactggcc cgcctgccca tgctccagac tctgcgtctg cagatgaact tcatcaacca 1320ggcccagctc ggcatcttca gggccttccc tggcctgcgc tacgtggacc tgtcggacaa 1380ccgcatcagc ggagcttcgg agctgacagc caccatgggg gaggcagatg gaggggagaa 1440ggtctggctg cagcctgggg accttgctcc ggccccagtg gacactocca gctctgaaga 1500cttcaggccc aactgcagca ccctcaactt caccttggat ctgtcacgga acaacctggt 1560gaccgtgcag ccggagatgt ttgcccagct ctcgcacctg cagtgcctgc gcctgagcca 1620caactgcatc tcgcaggcag tcaatggctc ccagttcctg ccgctgaccg gtctgcaggt 1680gctagacctg tcccacaata agctggacct ctaccacgag cactcattca cggagctacc 1740acgactggag gccctggacc tcagctacaa cagccagccc tttggcatgc agggcgtggg 1800ccacaacttc agcttcgtgg ctcacctgcg caccctgcgc cacctcagcc tggcccacaa 1860caacatccac agccaagtgt cccagcagct ctgcagtacg tcgctgcggg ccctggactt 1920cagcggcaat gcactgggcc atatgtgggc cgagggagac ctctatctgc acttcttcca 1980aggcctgagc ggtttgatct ggctggactt gtcccagaac cgcctgcaca ccctcctgcc 2040ccaaaccctg cgcaacctcc ccaagagcct acaggtgctg cgtctccgtg acaattacct 2100ggccttcttt aagtggtgga gcctccactt cctgcccaaa ctggaagtcc tcgacctggc 2160aggaaaccag ctgaaggccc tgaccaatgg cagcctgcct gctggcaccc ggctccggag 2220gctggatgtc agctgcaaca gcatcagctt cgtggccccc ggcttctttt ccaaggccaa 2280ggagctgcga gagctcaacc ttagcgccaa cgccctcaag acagtggacc actcctggtt 2340tgggcccctg gcgagtgccc tgcaaatact agatgtaagc gccaaccctc tgcactgcgc 2400ctgtggggcg gcctttatgg acttcctgct ggaggtgcag gctgccgtgc ccggtctgcc 2460cagccgggtg aagtgtggca gtccgggcca gctccagggc ctcagcatct ttgcacagga 2520cctgcgcctc tgCctggatg aggccctctc ctgggactgt ttcgccctct cgctgctggc 2580tgtggctctg ggcctgggtg tgCccatgct gcatcacctc tgtggctggg acctctggta 2640ctgcttccac Ctgtgcctgg cctggcttcc ctggcggggg cggcaaagtg ggcgagatga 2700ggatgccctg ccCtaCgatg ccttcgtggt cttcgacaaa acgcagagcg cagtggcaga 2760ctgggtgtaC aacgagcttc gggggcagct ggaggagtgc cgtgggcgct gggcactccg 2820cctgtgcctg gaggaacgcg actggctgcc tggcaaaacc ctctttgaga acctgtgggc 2880ctcggtctat ggcagccgca.agacgctgtt tgtgctggcc cacacggacc gggtcagtgg 2940tctcttgcgc gccagcttcc tgctggccca gcagcgcctg ctggaggacc gcaaggacgt 3000cgtggtgctg gtgatcctga gccctgacgg ccgccgctcc cgctacgtgc ggctgcgcca 3060gcgcctctgc cgccagagtg tcctcctctg gccccaccag cccagtggtc agcgcagctt 3120ctgggcccag ctgggcatgg ccctgaccag ggacaaccac cacttctata accggaactt 3180ctgccaggga cccacggccg aatagccgtg agccggaatc ctgcacggtg ccacctccac 3240actcacctca cctctgc 3258 SEQ ID NO:36     Human TLR9 amino acid (1055)MPMKWSGWRW SWGPATHTAL PPPQGFCRSA LHPLSLLVQA IMLAMTLALG TLPAFLPCEL 60QPHGLVNCNW LFLKSVPHFS MAAPRGNVTS LSLSSNRIHH LRDSDFAHLP SLRHLNLKWN 120CPPVGLSPMH FPCHMTIEPS TFLAVPTLEE LNLSYNNIMT VPALPKSLIS LSLSHTNILM 180LDSASLAGLH ALRFLFMDGN CYYKNPCRQA LEVAPGALLG LGNLTHLSLK YNNLTVVPRN 240LPSSLEYLLL SYNRIVKLAP EDLANLTALR VLDVGGNCRR CDHAPNPCME CPRHFPQLHP 300DTFSHLSRLE GLVLKDSSLS WLNASWFRGL GNLRVLDLSE NFLYKCITKT KAFQGLTQLR 360KLNLSFNYQK RVSFAHLSLA PSFGSLVALK ELDMHGIFFR SLDETTLRPL ARLPMLQTLR 420LQMNFINQAQ LGIFRAFPGL RYVDLSDNRI SGASELTATM GEADGGEKVW LQPGDLAPAP 480VDTPSSEDFR PNCSTLNFTL DLSRNNLVTV QPEMFAQLSH LQCLRLSHNC ISQAVNGSQF 540LPLTGLQVLD LSHNKLDLYH EHSFTELPRL EALDLSYNSQ PFGMQGVGHN FSFVAHLRTL 600RHLSLAHNNI HSQVSQQLCS TSLRALDFSG NALGHMWAEG DLYLHFFQGL SGLIWLDLSQ 660NRLHTLLPQT LRNLPKSLQV LRLRDNYLAF FKWWSLHFLP KLEVLDLAGN QLKALTNGSL 720TAGTRLRRLD VSCNSISFVA PGFFSKAKEL RELNLSANAL KTVDHSWFGP LASALQILDV 780SANPLHCACG AAFMDFLLEV QAAVPGLPSR VKCGSPGQLQ GLSIFAQDLR LCLDEALSWD 840CFALSLLAVA LGLGVPMLHH LCGWDLWYCF HLCLAWLPWR GRQSGRDEDA LPYDAFVVFD 900KTQSAVADWV YNELRGQLEE CRGRWALRLC LEERDWLPGK TLFENLWASV YGSRKTLFVL 960AHTDRVSGLL PASFLLAQQR LLEDRKDVVV LVILSPDGRR SRYVRLRQRL CRQSVLLWPH 1020QPSGQRSFWA QLGMALTRDN HHFYNRNFCQ GPTAE 1055SEQ ID NO:37     Human TLR9 nucleotideatgcccatga agtggagtgg gtggaggtgg agctgggggc cggccactca cacagccctc 60ccacccccac agggtttctg ccgcagcgcc ctgcacccgc tgtctctcct ggtgcaggcc 120atcatgctgg ccatgaccct ggccctgggt accttgcctg ccttcctacc ctgtgagctc 180cagccccacg gcctggtgaa ctgcaactgg ctgttcctga agtctgtgcc ccacttctcc 240atggcagcac cccgtggcaa tgtcaccagc ctttccttgt cctccaaccg catccaccac 300ctccatgatt ctgactttgc ccacctgccc agcctgcggc atctcaacct caagtggaac 360tgcccgccgg ttggcctcag ccccatgcac ttcccctgcc acatgaccat cgagcccagc 420accttcttgg ctgtgcccac cctggaagag ctaaacctga gctacaacaa catcatgact 480gtgcctgcgc tgcccaaatc cctcatatcc ctgtccctca gccataccaa catcctgatg 540ctagactctg ccagcctcgc cggcctgcat gccctgcgct tcctattcat ggacggcaac 600tgttattaca agaacccctg caggcaggca ctggaggtgg ccccgggtgc cctccttggc 660ctgggcaacc tcacccacct gtcactcaag tacaacaacc tcactgtggt gccccgcaac 720ctgccttcca gcctggagta tctgctgttg tcctacaacc gcatcgtcaa actggcgcct 780gaggacctgg ccaatctgac cgccctgcgt gtgctcgatg tgggcggaaa ttgccgccgc 840tgcgaccacg ctcccaaccc ctgcatggag tgccctcgtc acttccccca gctacatccc 900gataccttca gccacctgag ccgtcttgaa ggcctggtgt tgaaggacag ttctctctcc 960tggctgaatg ccagttggtt ccgtgggctg ggaaacctcc gagtgctgga cctgagtgag 1020aacttcctct acaaatgcat cactaaaacc aaggccttcc agggcctaac acagctgcgc 1080aagcttaacc tgtccttcaa ttaccaaaag agggtgtcct ttgcccacct gtctctggcc 1140ccttccttcg ggagcctggt cgccctgaag gagctggaca tgcacggcat cttcttccgc 1200tcactcgatg agaccacgct ccggccactg gcccgcctgc ccatgctcca gactctgcgt 1260ctgcagatga acttcatcaa ccaggcccag ctcggcatct tcagggcctt ccctggcctg 1320cgctacgtgg acctgtcgga caaccgcatc agcggagctt cggagctgac agccaccatg 1380ggggaggcag atggagggga gaaggtctgg ctgcagcctg gggaccttgc tccggcccca 1440gtggacactc ccagctctga agacttcagg cccaactgca gcaccctcaa cttcaccttg 1500gatctgtcac ggaacaacct ggtgaccgtg cagccggaga tgtttgccca gctctcgcac 1560ctgcagtgcc tgcgcctgag ccacaactgc atctcgcagg cagtcaatgg ctcccagttc 1620ctgccgctga ccggtctgca ggtgctagac ctgtcccaca ataagctgga cctctaccac 1680gagcactcat tcacggagct accacgactg gaggccctgg acctcagcta caacagccag 1720ccctttggca tgcagggcgt gggccacaac ttcagcttcg tggctcacct gcgcaccctg 1800cgccacctca gcctggccca caacaacatc cacagccaag tgtcccagca gctctgcagt 1860acgtcgctgc gggccctgga cttcagcggc aatgcactgg gccatatgtg ggccgaggga 1920gacctctatc tgcacttctt ccaaggcctg agcggtttga tctggctgga cttgtcccag 1980aaccgcctgc acaccctcct gccccaaacc ctgcgcaacc tccccaagag cctacaggtg 2040ctgcgtctcc gtgacaatta cctggccttc tttaagtggt ggagcctcca cttcctgccc 2100aaactggaag tcctcgacct ggcaggaaac cagctgaagg ccctgaccaa tggcagcctg 2160cctgctggca cccggctccg gaggctggat gtcagctgca acagcatcag cttcgtggcc 2220cccggcttct tttccaaggc caaggagctg cgagagctca accttagcgc caacgccctc 2280aagacagtgg accactcctg gtttgggccc ctggcgagtg ccctgcaaat actagatgta 2340agcgccaacc ctctgcactg cgcctgtggg gcggccttta tggacttcct gctggaggtg 2400caggctgccg tgcccggtct gcccagccgg gtgaagtgtg gcagtccggg ccagctccag 2460ggcctcagca tctttgcaca ggacctgcgc ctctgcctgg atgaggccct ctcctgggac 2520tgtttcgccc tctcgctgct ggctgtggct ctgggcctgg gtgtgcccat gctgcatcac 2580ctctgtggct gggacctctg gtactgcttc cacctgtgcc tggcctggct tccctggcgg 2640gggcggcaaa gtgggcgaga tgaggatgcc ctgccctacg atgccttcgt ggtcttcgac 2700aaaacgcaga gcgcagtggc agactgggtg tacaacgagc ttcgggggca gctggaggag 2760tgccgtgggc gctgggcact ccgcctgtgc ctggaggaac gcgactggct gcctggcaaa 2820accctctttg agaacctgtg ggcctcggtc tatggcagcc gcaagacgct gtttgtgctg 2880gcccacacgg accgggtcag tggtctcttg cgcgccagct tcctgctggc ccagcagcgc 2940ctgctggagg accgcaagga cgtcgtggtg ctggtgatcc tgagccctga cggccgccgc 3000tcccgctatg tgcggctgcg ccagcgcctc tgccgccaga gtgtcctcct ctggccccac 3060cagcccagtg gtcagcgcag cttctgggcc cagctgggca tggccctgac cagggacaac 3120caccacttct ataaccggaa cttctgccag ggacccacgg ccgaa 3165SEQ ID NO:38     Murine TLR9 amino acidMVLRRRTLHP LSLLVQAAVL AETLALGTLP AFLPCELKPH GLVDCNWLFL KSVPRFSAAA 60SCSNITRLSL ISNRIHHLHN SDFVHLSNLR QLNLKWNCPP TGLSPLHFSC HMTIEPRTFL 120AMRTLEELNL SYNGITTVPR LPSSLVNLSL SRTNILVLDA NSLAGLYSLR VLFMDGNCYY 180KNPCTGAVKV TPGALLGLSN LTHLSLKYNN LTKVPRQLPP SLEYLLVSYN LIVKLGPEDL 240ANLTSLRVLD VGGNCRRCDH APHPCIECGQ KSLHLHPETF HHLSHLEGLV LKDSSLHTLN 300SSWFQGLVNL SVLDLSENFL YESINHTNAF QNLTRLRKLN LSFNYRKKVS FARLHLASSF 360KNLVSLQELN MNGIFFRSLN KYTLRWLADL PKLHTLHLQM NFINQAQLSI FGTFRALRFV 420DLSDNRISGP STLSEATPEE ADDAEQEELL SADPHPAPLS TPASKNFMDR CKNFKFTMDL 480SRNNLVTIKP EMFVNLSRLQ CLSLSHNSIA QAVNGSQFLP LTNLQVLDLS HNKLDLYHWK 540SFSELPQLQA LDLSYNSQPF SMKGIGHNFS FVAHLSMLHS LSLAHNDIHT RVSSHLNSNS 600VRFLDFSGNG MGRMWDEGGL YLHFFQGLSG LLKLDLSQNN LHILRPQNLD NLPKSLKLLS 660LRDNYLSFFN WTSLSFLPNL EVLDLAGNQL KALTNGTLPN GTLLQKLDVS SNSIVSVVPA 720FFALAVELKE VNLSHNILKT VDRSWFGPIV NMLTVLDVRS NPLHCACGAA FVDLLLEVQT 780KVPGLANGVK CGSPGQLQGR SIFAQDLRLC LDEVLSWDCF GLSLLAVAVG MVVPILHHLC 840GWDVWYCFHL CLAWLPLLAR SRRSAQALPY DAFVVFDKAQ SAVADWVYNE LRVRLEERRG 900RRALRLCLED RDWLPGQTLF ENLWASIYGS RKTLFVLAHT DRVSGLLRTS FLLAQQRLLE 960DRKDVVVLVI LRPDAHRSRY VRLRQRLCRQ SVLFWPQQPN GQGGFWAQLS TALTRDNRHF 1020YNQNFCRGPT AE 1032 SEQ ID NO:39     Murine TLR9 nucleotidetgtcagaggg agcctcggga gaatcctcca tctcccaaca tggttctccg tcgaaggact 60ctgcacccct tgtccctcct ggtacaggct gcagtgctgg ctgagactct ggccctgggt 120accctgcctg ccttcctacc ctgtgagctg aagcCtCatg gcctggtgga ctgcaattgg 180ctgttcctga agtctgtacc ccgtttctct gcggcagcat cctgctccaa catcacccgc 240ctctccttga tctccaaccg tatccaccac ctgcacaact ccgacttcgt ccacctgtcc 300aacctgcggc agctgaacct caagtggaac tgtccaccca ctggccttag ccccctgcac 360ttctcttgcc acatgaccat tgagcccaga accttcctgg ctatgcgtac actggaggag 420ctgaacctga gctataatgg tatcaccact gtgccccgac tgcccagctc cctggtgaat 480ctgagcctga gccacaccaa catcctggtt ctagatgcta acagcctcgc cggcctatac 540agcctgcgcg ttctcttcat ggacgggaac tgctactaca agaacccctg cacaggagcg 600gtgaaggtga ccccaggcgc cctcctgggc ctgagcaatc tcacccatct gtctctgaag 660tataacaacc tcacaaaggt gccccgccaa ctgcccccca gcctggagta cctcctggtg 720tcctataacc tcattgtcaa gctggggcct gaagacctgg ccaatctgac ctcccttcga 780gtacttgatg tgggtgggaa ttgccgtcgc tgcgaccatg cccccaatcc ctgtatagaa 840tgtggccaaa agtccctcca cctgcaccct gagaccttcc atcacctgag ccatctggaa 900ggcctggtgc tgaaggacag ctctctdcat acactgaact cttcctggtt ccaaggtctg 960gtcaacctct cggtgctgga cctaagcgag aactttctct atgaaagcat caaccacacc 1020aatgcctttc agaacctaac ccgcctgcgc aagctcaacc tgtccttcaa ttaccgcaag 1080aaggtatcct ttgcccgcct ccacctggca agttccttca agaacctggt gtcactgcag 1140gagctgaaca tgaacggcat cttcttccgc tcgctcaaca agtacacgct cagatggctg 1200gccgatctgc ccaaactcca cactctgcat cttcaaatga acttcatcaa ccaggcacag 1260ctcagcatct ttggtacctt ccgagccctt-cgctttgtgg acttgtcaga caatcgcatc 1320agtgggcctt caacgctgtc agaagccacc cctgaagagg cagatgatgc agagcaggag 1380gagctgttgt ctgcggatcc tcacccagct ccactgagca cccctgcttc taagaacttc 1440atggacaggt gtaagaactt caagttcacc atggacctgt ctcggaacaa cctggtgact 1500atcaagccag agatgtttgt caatctctca cgcctccagt gtcttagcct gagccacaac 1560tccattgcac aggctgtcaa tggctctcag ttcctgccgc tgactaatct gcaggtgctg 1620gacctgtccc ataacaaact ggacttgtac cactggaaat cgttcagtga gctaccacag 1680ttgcaggccc tggacctgag ctacaacagc cagcccttta gcatgaaggg tataggccac 1740aatttcagtt ttgtggccca tctgtccatg ctacacagcc ttagcctggc acacaatgac 1800attcataccc gtgtgtcctc acatctcaac agcaactcag tgaggtttct tgacttcagc 1860ggcaacggta tgggccgcat gtgggatgag gggggccttt atctccattt cttccaaggc 1920ctgagtggcc tgctgaagct ggacctgtct caaaataacc tgcatatcct ccggccccag 1980aaccttgaca acctccccaa gagcctgaag ctgctgagcc tccgagacaa ctacctatct 2040ttctttaact ggaccagtct gtccttcctg cccaacctgg aagtcctaga cctggcaggc 2100aaccagctaa aggccctgac caatggcacc ctgcctaatg gcaccctcct ccagaaactg 2160gatgtcagca gcaacagtat cgtctctgtg gtcccagcct tcttcgctct ggcggtcgag 2220ctgaaagagg tcaacctcag ccacaacatt ctcaagacgg tggatcgctc ctggtttggg 2280cccattgtga tgaacctgac agttctagac gtgagaagca accctctgca ctgtgcctgt 2340ggggcagcct tcgtagactt actgttggag gtgcagacca aggtgcctgg cctggctaat 2400ggtgtgaagt gtggcagccc cggccagctg cagggccgta gcatcttcgc acaggacctg 2460cggctgtgcc tggatgaggt cctctcttgg gactgctttg gcctttcact cttggctgtg 2520gccgtgggca tggtggtgcc tatactgcac catctctgcg gctgggacgt ctggtactgt 2580tttcatctgt gcctggcatg gctacctttg ctggcccgca gccgacgcag cgcccaagct 2640ctcccctatg atgccttcgt ggtgttcgat aaggcacaga gcgcagttgc ggactgggtg 2700tataacgagc tgcgggtgcg gctggaggag cggcgcggtc gccgagccct acgcttgtgt 2760ctggaggacc gagattggct gcctggccag acgctcttcg agaacctctg ggcttccatc 2820tatgggagcc gcaagactct atttgtgctg gcccacacgg accgcgtcag tggcctcctg 2880cgcaccagct tcctgctggc tcagcagcgc ctgttggaag accgcaagga cgtggtggtg 2940ttggtgatcc tgcgtccgga tgcccaccgc tcccgctatg tgcgactgcg ccagcgtctc 3000tgccgccaga gtgtgctctt ctggccccag cagcccaacg ggcagggggg cttctgggcc 3060cagctgagta cagccctgac tagggacaac cgccacttct ataaccagaa cttctgccgg 3120ggacctacag cagaatagct cagagcaaca gctggaaaca gctgcatctt catgcctggt 3180tcccgagttg ctctgcctgc 3200

Ribonucleoside vanadyl complexes (i.e., mixtures of adenine, cytosine,guanosine, and uracil ribonucleoside vanadyl complexes), are well knownby those of skill in the art as RNAse inhibitors. Berger S L et al.(1979) Biochemistry 18:5143; Puskas R S et al. (1982) Biochemistry21:4602. Ribonucleoside vanadyl complexes are commercially availablefrom suppliers including Sigma-Aldrich, Inc.

In one embodiment, the immunostimulatory G,U-containing RNA oligomer ofthe invention does not contain a CpG dinucleotide and is not a CpGimmunostimulatory nucleic acid. In some embodiments, a CpGimmunostimulatory nucleic acid is used in the methods of the invention.

A CpG immunostimulatory nucleic acid is a nucleic acid which contains aCG dinucleotide, the C residue of which is unmethylated. CpGimmunostimulatory nucleic acids are known to stimulate Th1-type immuneresponses. CpG sequences, while relatively rare in human DNA arecommonly found in the DNA of infectious organisms such as bacteria. Thehuman immune system has apparently evolved to recognize CpG sequences asan early warning sign of infection and to initiate an immediate andpowerful immune response against invading pathogens without causingadverse reactions frequently seen with other immune stimulatory agents.Thus CpG containing nucleic acids, relying on this innate immune defensemechanism can utilize a unique and natural pathway for immune therapy.The effects of CpG nucleic acids on immune modulation have beendescribed extensively in U.S. patents such as U.S. Pat. Nos. 6,194,388B1, 6,207,646 B1, 6,239,116 B1 and 6,218,371 B1, and published patentapplications, such as PCT/US98/03678, PCT/US98/10408, PCT/US98/04703,and PCT/US99/09863. The entire contents of each of these patents andpatent applications is hereby incorporated by reference.

A CpG nucleic acid is a nucleic acid which includes at least oneunmethylated CpG dinucleotide. A nucleic acid containing at least oneunmethylated CpG dinucleotide is a nucleic acid molecule which containsan unmethylated cytosine in a cytosine-guanine dinucleotide sequence(i.e., “CpG DNA” or DNA containing a 5′ cytosine followed by 3′guanosine and linked by a phosphate bond) and activates the immunesystem. The CpG nucleic acids can be double-stranded or single-stranded.Generally, double-stranded molecules are more stable in vivo, whilesingle-stranded molecules have increased immune activity. Thus in someaspects of the invention it is preferred that the nucleic acid be singlestranded and in other aspects it is preferred that the nucleic acid bedouble stranded. In certain embodiments, while the nucleic acid issingle stranded, it is capable of forming secondary and tertiarystructures (e.g., by folding back on itself, or by hybridizing withitself either throughout its entirety or at select segments along itslength). Accordingly, while the primary structure of such a nucleic acidmay be single stranded, its higher order structures may be double ortriple stranded. The terms CpG nucleic acid or CpG oligonucleotide asused herein refer to an immunostimulatory CpG nucleic acid unlessotherwise indicated. The entire immunostimulatory nucleic acid can beunmethylated or portions may be unmethylated but at least the C of the5′ CG 3′ must be unmethylated.

In one aspect the invention provides a method of activating an immunecell. The method involves contacting an immune cell with animmunostimulatory composition of the invention, described above, in aneffective amount to induce activation of the immune cell. As usedherein, an “immune cell” is cell that belongs to the immune system.Immune cells participate in the regulation and execution of inflammatoryand immune responses. They include, without limitation, B lymphocytes (Bcells), T lymphocytes (T cells), natural killer (NK) cells, dendriticcells, other tissue-specific antigen-presenting cells (e.g., Langerhanscells), macrophages, monocytes, granulocytes (neutrophils, eosinophils,basophils), and mast cells. Splenocytes, thymocytes, and peripheralblood mononuclear cells (PBMCs) include immune cells. Immune cells canbe isolated from the blood, spleen, marrow, lymph nodes, thymus, andother tissues using methods well known to those of skill in the art.Immune cells can also include certain cell lines as well as primarycultures maintained in vitro or ex vivo.

In one embodiment the activation of the immune cell involves secretionof a cytokine by the immune cell. In one embodiment the activation ofthe immune cell involves secretion of a chemokine by the immune cell. Inone embodiment the activation of the immune cell involves expression ofa costimulatory/accessory molecule by the immune cell. In one embodimentthe costimulatory/accessory molecule is selected from the groupconsisting of intercellular adhesion molecules (ICAMs, e.g., CD54),leukocyte function-associated antigens (LFAs, e.g., CD58), B7s (CD80,CD86), and CD40.

“Activation of an immune cell” shall refer to a transition of an immunecell from a resting or quiescent state to a state of heightenedmetabolic activity and phenotype associated with immune cell function.Such immune cell function can include, for example, secretion of solubleproducts such as immunoglobulins, cytokines, and chemokines; cellsurface expression of costimulatory/accessory molecules and MHCantigens; immune cell migration; phagocytosis and cytotoxic activitytoward target cells; and immune cell maturation. In some instancesimmune activation can refer to Th1 immune activation; in other instancesimmune activation can refer to Th2 immune activation.

“Th1 immune activation” as used herein refers to the activation ofimmune cells to express Th1-like secreted products, including certaincytokines, chemokines, and subclasses of immunoglobulin; and activationof certain immune cells. Th1-like secreted products include, forexample, the cytokines IFN-γ, IL-2, IL-12, IL-18, TNF-α, and thechemokine IP-10 (CXCL10). In the mouse, Th1 immune activation stimulatessecretion of IgG2a. Th1 immune activation also may include activation ofNK cells and dendritic cells, i.e., cells involved in cellular immunity.Th1 immune activation is believed to counter-regulate Th2 immuneactivation.

“Th2 immune activation” as used herein refers to the activation ofimmune cells to express Th2-like secreted products, including certaincytokines and subclasses of immunoglobulin. Th2-like secreted productsinclude, for example, the cytokines IL-4 and IL-10. In the mouse, Th2immune activation stimulates secretion of IgG1 and IgE. Th2 immuneactivation is believed to counter-regulate Th1 immune activation.

In another aspect, the invention provides a method of inducing an immuneresponse in a subject. The method entails administering to a subject acomposition of the invention in an effective amount to induce an immuneresponse in the subject. Thus the compositions of the invention may beused to treat a subject in need of immune activation. A subject in needof immune activation may include a subject in need of Th1-like immuneactivation.

The compositions and methods of the invention can be used, alone or inconjunction with other agents, to treat a subject in need of Th1-likeimmune activation. A “subject in need of Th1-like immune activation” isa subject that has or is at risk of developing a disease, disorder, orcondition that would benefit from an immune response skewed toward Th1.Such a subject may have or be at risk of having a Th2-mediated disorderthat is susceptible to Th1-mediated cross-regulation or suppression.Such disorders include, for example, certain organ-specific autoimmunediseases. Alternatively, such a subject may have or be at risk of havinga Th1-deficient state. Such disorders include, for example, tumors,infections with intracellular pathogens, and AIDS.

As used herein, “G,U-rich RNA” shall mean RNA at least 5 nucleotideslong that by base composition is at least 60 percent, more preferably atleast 80 percent, and most preferably at least 90 percent guanine (G)and uracil (U). Such base composition is measured over the full lengthof the RNA if it is no more than 10 bases long, and over a stretch of atleast 10 contiguous bases if the RNA is more than 10 bases long.

As used herein, “G-rich RNA” shall mean RNA that by base composition isat least 70 percent, more preferably at least 80 percent, even morepreferably at least 90 percent, and most preferably at least 95 percentguanine (G). Such base composition is measured over the full length ofthe RNA if it is no more than 10 bases long, and over a stretch of atleast 10 contiguous bases if the RNA is more than 10 bases long.

In some embodiments the compositions of the present invention include aDNA:RNA conjugate. A DNA:RNA conjugate shall mean a molecule or complexthat includes at least one deoxyribonucleoside linked to at least oneribonucleoside. The deoxyribonucleoside and ribonucleoside componentsmay be linked by base pair interaction. Alternatively, thedeoxyribonucleoside and ribonucleoside components may be linked bycovalent linkage between the sugar moieties of the at least onedeoxyribonucleoside and the at least one ribonucleoside. The covalentlinkage between the sugar moieties may be direct or indirect, forexample through a linker. Base pair interactions typically are, but arenot limited to, non-covalent Watson-Crick type base pair interactions.Other base pair interactions, including non-covalent (e.g., Hoogsteinbase pairing) and covalent interactions are contemplated by theinvention. Base pair interactions also typically will involve duplexformation involving two strands, but higher order interactions are alsocontemplated by the invention.

A DNA:RNA conjugate involving a covalent linkage between the sugarmoieties of the at least one deoxyribonucleoside and the at least oneribonucleoside is referred to herein as having a chimeric DNA:RNAbackbone. The DNA:RNA conjugate having a chimeric DNA:RNA backbone willhave primary structure defined by its base sequence, and it may furtherhave a secondary or higher order structure. A secondary or higher orderstructure will include at least one intramolecular base pairinteraction, e.g., a stem-loop structure, or intermolecular base pairinteraction.

Heteroduplex base pairing shall refer to intramolecular orintermolecular base pair interaction between DNA and RNA. For example,heteroduplex base pairing may occur between individual complementarysingle-stranded DNA and RNA molecules. Alternatively, as in the case ofsuitable DNA:RNA chimeric backbone nucleic acid molecules, heteroduplexbase pairing may occur between complementary DNA and RNA regions withinthe same molecule.

In some embodiments the compositions of the present invention include achimeric DNA:RNA backbone having a cleavage site between the DNA andRNA. A cleavage site refers to a structural element along the chimericbackbone that is susceptible to cleavage by any suitable means. Thecleavage site may be a phosphodiester bond that is relativelysusceptible to cleavage by endonuclease. In this instance the DNA andRNA each may include internucleotide linkages that are stabilized, suchthat the chimeric backbone is most susceptible to endonuclease cleavageat the phosphodiester junction between the stabilized DNA and thestabilized RNA. The cleavage site may be designed so that it issusceptible to cleavage under certain pH conditions, e.g., relativelymore stable at higher pH than at lower pH, or vice versa. Such pHsensitivity may be accomplished, for example, by preparation of thechimeric DNA:RNA composition in liposomes. The cleavage site may involvea disulfide linkage. Such disulfide linkage may be relatively morestable under oxidizing conditions than under reducing conditions, e.g.,the latter conditions present within an endosome. The cleavage site mayalso involve a linker that is susceptible to cleavage by an enzyme, pH,redox condition, or the like. In some embodiments the composition mayinclude more than one cleavage site.

Conjugates of the invention permit selection of fixed molar ratios ofthe components of the conjugates. In the case of DNA:RNA conjugates itmay be advantageous or convenient to have a 1:1 ratio of DNA and RNA.Conjugates that are heteroduplex DNA:RNA will commonly have a 1:1 ratioof DNA and RNA. Conjugates that have a chimeric DNA:RNA backbone mayalso commonly have a 1:1 ratio of DNA and RNA. Conjugates having otherDNA:RNA ratios are contemplated by the invention, including, but notlimited to, 1:2, 1:3, 1:4, 2:1, 3:1, 4:1, and so on. The conjugation maystabilize one or more components in comparison to the stability of thesame component or components alone. Conjugatation may also facilitatedelivery of the components into cells at the selected ratio.

Cleavage sites may serve any of several purposes useful in the presentinvention. Once delivered to a cell of interest, the components joinedvia the cleavage site (or sites) may be liberated to becomeindependently or optimally active within the cell or in the vicinity ofthe cell. In some embodiments the cleavage sites may be important topharmacokinetics of at least one of the components of the conjugate. Forinstance, the cleavage sites may be designed and selected to confer anextended time release of one of the components.

The invention generally provides efficient methods of identifyingimmunostimulatory compounds and the compounds and agents so identified.Generally, the screening methods involve assaying for compounds whichinhibit or enhance signaling through a particular TLR. The methodsemploy a TLR, a suitable reference ligand for the TLR, and a candidateimmunostimulatory compound. The selected TLR is contacted with asuitable refernce compound (TLR ligand) and a TLR-mediated referencesignal is measured. The selected TLR is also contacted with a candidateimmunostimulatory compound and a TLR-mediated test signal is measured.The test signal and the reference signal are then compared. A favorablecandidate immunostimulatory compound may subsequently be used as areference compound in the assay. Such methods are adaptable toautomated, high throughput screening of candidate compounds. Examples ofsuch high throughput screening methods are described in U.S. Pat. Nos.6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158;5,443,791; 5,429,921; and 5,143,854.

The assay mixture comprises a candidate immunostimulatory compound.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateimmunostimulatory compounds encompass numerous chemical classes,although typically they are organic compounds. Preferably, the candidateimmunostimulatory compounds are small organic compounds, i.e., thosehaving a molecular weight of more than 50 yet less than about 2500.Polymeric candidate immunostimulatory compounds can have highermolecular weights, e.g., oligonucleotides in the range of about 2500 toabout 12,500. Candidate immunostimulatory compounds comprise functionalchemical groups necessary for structural interactions with polypeptides,and may include at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups and morepreferably at least three of the functional chemical groups. Thecandidate immunostimulatory compounds can comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one or more of the above-identified functional groups.Candidate immunostimulatory compounds also can be biomolecules such asnucleic acids, peptides, saccharides, fatty acids, sterols, isoprenoids,purines, pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the candidate immunostimulatorycompound is a nucleic acid, the candidate immunostimulatory compoundtypically is a DNA or RNA molecule, although modified nucleic acidshaving non-natural bonds or subunits are also contemplated.

Candidate immunostimulatory compounds are obtained from a wide varietyof sources, including libraries of natural, synthetic, or semisyntheticcompounds, or any combination thereof. For example, numerous means areavailable for random and directed synthesis of a wide variety of organiccompounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs of the candidate immunostimulatory compounds.

Therefore, a source of candidate immunostimulatory compounds arelibraries of molecules based on known TLR ligands, e.g., CpGoligonucleotides known to interact with TLR9, in which the structure ofthe ligand is changed at one or more positions of the molecule tocontain more or fewer chemical moieties or different chemical moieties.The structural changes made to the molecules in creating the librariesof analog inhibitors can be directed, random, or a combination of bothdirected and random substitutions and/or additions. One of ordinaryskill in the art in the preparation of combinatorial libraries canreadily prepare such libraries based on existing TLR9 ligands.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The order of addition of components, incubation temperature, time ofincubation, and other parameters of the assay may be readily determined.Such experimentation merely involves optimization of the assayparameters, not the fundamental composition of the assay. Incubationtemperatures typically are between 4° C. and 40° C. Incubation timespreferably are minimized to facilitate rapid, high throughput screening,and typically are between 1 minute and 10 hours.

After incubation, the level of TLR signaling is detected by anyconvenient method available to the user. For cell-free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. For example, separation can be accomplished in solution, or,conveniently, at least one of the components is immobilized on a solidsubstrate, from which the unbound components may be easily separated.The solid substrate can be made of a wide variety of materials and in awide variety of shapes, e.g., microtiter plate, microbead, dipstick,resin particle, etc. The substrate preferably is chosen to maximizesignal-to-noise ratios, primarily to minimize background binding, aswell as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatographic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as measurement of an induced polypeptide within, on the surface of,or secreted by the cell. Examples of detection methods useful incell-based assays include fluorescence-activated cell sorting (FACS)analysis, bioluminescence, fluorescence, enzyme-linked immunosorbentassay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR),and the like. Examples of detection methods useful in cell-free assaysinclude bioluminescence, fluorescence, enzyme-linked immunosorbent assay(ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), andthe like.

A subject shall mean a human or animal including but not limited to adog, cat, horse, cow, pig, sheep, goat, chicken, rodent, e.g., rats andmice, primate, e.g., monkey, and fish or aquaculture species such as finfish (e.g., salmon) and shellfish (e.g., shrimp and scallops). Subjectssuitable for therapeutic or prophylactic methods include vertebrate andinvertebrate species. Subjects can be house pets (e.g., dogs, cats,fish, etc.), agricultural stock animals (e.g., cows, horses, pigs,chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.),zoo animals (e.g., lions, giraffes, etc.), but are not so limited.Although many of the embodiments described herein relate to humandisorders, the invention is also useful for treating other nonhumanvertebrates.

As used herein, the term “treat”, when used with respect to one of thedisorders described herein, refers both to a prophylactic treatmentwhich decreases the likelihood that a subject will develop the disorderas well as to treatment of an established disorder, e.g., to reduce oreliminate the disorder or symptoms of the disorder, or to prevent thedisorder or symptoms of the disorder from becoming worse.

A subject that has a disorder refers to a subject that has anobjectively measureable manifestation of the disorder. Thus for examplea subject that has a cancer is a subject that has detectable cancerouscells. A subject that has an infection is a subject that has beenexposed to an infectious organism and has acute or chronic detectablelevels of the organism in the body. The infection may be latent(dormant) or active.

A subject at risk of having a disorder is defined as a subject that hasa higher than normal risk of developing the disorder. The normal risk isgenerally the risk of a population of normal individuals that do nothave the disorder and that are not identifiably predisposed, e.g.,either genetically or environmentally, to developing the disorder. Thusa subject at risk of having a disorder may include, without limitation,a subject that is genetically predisposed to developing the disorder, aswell as a subject that is or will be exposed to an environmental agentknown or believed to cause the disorder. Environmental agentsspecifically include, but are not limited to, infectious agents such asviruses, bacteria, fungi, and parasites. Other environmental agents mayinclude, for example, tobacco smoke, certain organic chemicals,asbestos, and the like.

The term “effective amount” of a nucleic acid or other therapeutic agentrefers to the amount necessary or sufficient to realize a desiredbiologic effect. In general, an effective amount is that amountnecessary to cause activation of the immune system, resultingpotentially in the development of an antigen-specific immune response.In some embodiments, the nucleic acid or other therapeutic agent areadministered in an effective amount to stimulate or induce a Th1 immuneresponse or a general immune response. An effective amount to stimulatea Th1 immune response may be defined as that amount which stimulates theproduction of one or more Th1-type cytokines, such as IL-2, IL-12,TNF-α, and IFN-γ, and/or production of one or more Th1-type antibodies.

In yet another aspect the invention provides a method of inducing animmune response in a subject. The method according to this aspect of theinvention involves administering to a subject an antigen, andadministering to the subject an immunostimulatory composition of theinvention in an effective amount to induce an immune response to theantigen. It is to be noted that the antigen may be administered before,after, or concurrently with the immunostimulatory composition of theinvention. In addition, both the antigen and the immunostimulatorycompound can be administered to the subject more than once.

The invention further provides, in yet another aspect, a method ofinducing an immune response in a subject. The method according to thisaspect of the invention involves isolating dendritic cells of a subject,contacting the dendritic cells ex vivo with an immunostimulatorycomposition of the invention, contacting the dendritic cells ex vivowith an antigen, and administering the contacted dendritic cells to thesubject.

The term “antigen” refers to a molecule capable of provoking an immuneresponse. The term antigen broadly includes any type of molecule that isrecognized by a host system as being foreign. Antigens include but arenot limited to microbial antigens, cancer antigens, and allergens.Antigens include, but are not limited to, cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, and carbohydrates. Manyantigens are protein or polypeptide in nature, as proteins andpolypeptides are generally more antigenic than carbohydrates or fats.

The antigen may be an antigen that is encoded by a nucleic acid vectoror it may not be encoded in a nucleic acid vector. In the former casethe nucleic acid vector is administered to the subject and the antigenis expressed in vivo. In the latter case the antigen may be administereddirectly to the subject. An antigen not encoded in a nucleic acid vectoras used herein refers to any type of antigen that is not a nucleic acid.For instance, in some aspects of the invention the antigen not encodedin a nucleic acid vector is a peptide or a polypeptide. Minormodifications of the primary amino acid sequences of peptide orpolypeptide antigens may also result in a polypeptide which hassubstantially equivalent antigenic activity as compared to theunmodified counterpart polypeptide. Such modifications may bedeliberate, as by site-directed mutagenesis, or may be spontaneous. Allof the polypeptides produced by these modifications are included hereinas long as antigenicity still exists. The peptide or polypeptide may be,for example, virally derived. The antigens useful in the invention maybe any length, ranging from small peptide fragments of a full lengthprotein or polypeptide to the full length form. For example, the antigenmay be less than 5, less than 8, less than 10, less than 15, less than20, less than 30, less than 50, less than 70, less than 100, or moreamino acid residues in length, provided it stimulates a specific immuneresponse.

The nucleic acid encoding the antigen is operatively linked to a geneexpression sequence which directs the expression of the antigen nucleicacid within a eukaryotic cell. The gene expression sequence is anyregulatory nucleotide sequence, such as a promoter sequence orpromoter-enhancer combination, which facilitates the efficienttranscription and translation of the antigen nucleic acid to which it isoperatively linked. The gene expression sequence may, for example, be amammalian or viral promoter, such as a constitutive or induciblepromoter. Constitutive mammalian promoters include, but are not limitedto, the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actinpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, the long terminal repeats (LTR) of Moloney leukemia virusand other retroviruses, and the thymidine kinase promoter of herpessimplex virus. Other constitutive promoters are known to those ofordinary skill in the art. The promoters useful as gene expressionsequences of the invention also include inducible promoters. Induciblepromoters are expressed in the presence of an inducing agent. Forexample, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined antigen nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

The antigen nucleic acid is operatively linked to the gene expressionsequence. As used herein, the antigen nucleic acid sequence and the geneexpression sequence are said to be operably linked when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the antigen coding sequence underthe influence or control of the gene expression sequence. Two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ gene expression sequence results in the transcription of theantigen sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the antigen sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to anantigen nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that antigen nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

The antigen nucleic acid of the invention may be delivered to the immunesystem alone or in association with a vector. In its broadest sense, avector is any vehicle capable of facilitating the transfer of theantigen nucleic acid to the cells of the immune system so that theantigen can be expressed and presented on the surface of the immunecell. The vector generally transports the nucleic acid to the immunecells with reduced degradation relative to the extent of degradationthat would result in the absence of the vector. The vector optionallyincludes the above-described gene expression sequence to enhanceexpression of the antigen nucleic acid in immune cells. In general, thevectors useful in the invention include, but are not limited to,plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the antigen nucleic acid sequences. Viral vectors are apreferred type of vector and include, but are not limited to, nucleicacid sequences from the following viruses: retrovirus, such as Moloneymurine leukemia virus, Harvey murine sarcoma virus, murine mammary tumorvirus, and Rous sarcoma virus; adenovirus, adeno-associated virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown in the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W. H.Freeman Co., New York (1990) and Murray, E. J. Methods in MolecularBiology, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

A preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus can beengineered to be replication-deficient and is capable of infecting awide range of cell types and species. It further has advantages, such asheat and lipid solvent stability; high transduction frequencies in cellsof diverse lineages, including hemopoietic cells; and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. Reportedly, wild-type adeno-associated virus manifestsome preference for integration sites into human cellular DNA, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion. Recombinantadeno-associated viruses that lack the replicase protein apparently lackthis integration sequence specificity.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operatively encoded within the plasmid.Some commonly used plasmids include pBR322, pUC18, pUC19, pRc/CMV, SV40,and pBlueScript. Other plasmids are well-known to those of ordinaryskill in the art. Additionally, plasmids may be custom designed usingrestriction enzymes and ligation reactions to remove and add specificfragments of DNA.

It has recently been discovered that gene-carrying plasmids can bedelivered to the immune system using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to immune cells, e.g., B cells,dendritic cells, likely by passing through the gut barrier. High levelsof immune protection have been established using this methodology. Suchmethods of delivery are useful for the aspects of the inventionutilizing systemic delivery of antigen, nucleic acids, and/or othertherapeutic agent.

In some aspects of the invention, the nucleic acids are administeredalong with therapeutic agents such as disorder-specific medicaments. Asused herein, a disorder-specific medicament is a therapy or agent thatis used predominately in the treatment or prevention of a disorder.

In one aspect, the combination of nucleic acid and disorder-specificmedicaments allows for the administration of higher doses ofdisorder-specific medicaments without as many side effects as areordinarily experienced at those high doses. In another aspect, thecombination of nucleic acid and disorder-specific medicaments allows forthe administration of lower, sub-therapeutic doses of either compound,but with higher efficacy than would otherwise be achieved using such lowdoses. As one example, by administering a combination of animmunostimulatory nucleic acid and a medicament, it is possible toachieve an effective response even though the medicament is administeredat a dose which alone would not provide a therapeutic benefit (i.e., asub-therapeutic dose). As another example, the combined administrationachieves a response even though the nucleic acid is administered at adose which alone would not provide a therapeutic benefit.

The nucleic acids and/or other therapeutic agents can also beadministered on fixed schedules or in different temporal relationshipsto one another. The various combinations have many advantages over theprior art methods of modulating immune responses or preventing ortreating disorders, particularly with regard to decreased non-specifictoxicity to normal tissues.

Cancer is a disease which involves the uncontrolled growth (i.e.,division) of cells. Some of the known mechanisms which contribute to theuncontrolled proliferation of cancer cells include growth factorindependence, failure to detect genomic mutation, and inappropriate cellsignaling. The ability of cancer cells to ignore normal growth controlsmay result in an increased rate of proliferation. Although the causes ofcancer have not been firmly established, there are some factors known tocontribute, or at least predispose a subject, to cancer. Such factorsinclude particular genetic mutations (e.g., BRCA gene mutation forbreast cancer, APC for colon cancer), exposure to suspectedcancer-causing agents, or carcinogens (e.g., asbestos, UV radiation) andfamilial disposition for particular cancers such as breast cancer.

The cancer may be a malignant or non-malignant cancer. Cancers or tumorsinclude but are not limited to biliary tract cancer; brain cancer;breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g., small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

A “subject having a cancer” is a subject that has detectable cancerouscells.

A “subject at risk of developing a cancer” is one who has a higher thannormal probability of developing cancer. These subjects include, forinstance, subjects having a genetic abnormality that has beendemonstrated to be associated with a higher likelihood of developing acancer, subjects having a familial disposition to cancer, subjectsexposed to cancer-causing agents (i.e., carcinogens) such as tobacco,asbestos, or other chemical toxins, and subjects previously treated forcancer and in apparent remission.

A “cancer antigen” as used herein is a compound, such as a peptide orprotein, associated with a tumor or cancer cell surface and which iscapable of provoking an immune response when expressed on the surface ofan antigen presenting cell in the context of an MHC molecule. Cancerantigens can be prepared from cancer cells either by preparing crudeextracts of cancer cells, for example, as described in Cohen P A et al.(1994) Cancer Res 54:1055-8, by partially purifying the antigens, byrecombinant technology, or by de novo synthesis of known antigens.Cancer antigens include but are not limited to antigens that arerecombinantly expressed, an immunogenic portion of, or a whole tumor orcancer. Such antigens can be isolated or prepared recombinantly or byany other means known in the art.

The terms “cancer antigen” and “tumor antigen” are used interchangeablyand refer to antigens which are differentially expressed by cancer cellsand can thereby be exploited in order to target cancer cells. Cancerantigens are antigens which can potentially stimulate apparentlytumor-specific immune responses. Some of these antigens are encoded,although not necessarily expressed, by normal cells. These antigens canbe characterized as those which are normally silent (i.e., notexpressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses. Examples of tumor antigens include MAGE,MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)—C017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100^(Pmel117), PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

Cancers or tumors and tumor antigens associated with such tumors (butnot exclusively), include acute lymphoblastic leukemia (etv6; aml1;cyclophilin b), B cell lymphoma (Ig-idiotype), glioma (E-cadherin;α-catenin; β-catenin; γ-catenin; p120ctn), bladder cancer (p21ras),biliary cancer (p21ras), breast cancer (MUC family; HER2/neu; c-erbB-2),cervical carcinoma (p53; p21ras), colon carcinoma (p21ras; HER2/neu;c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen(CRC)—C017-1A/GA733; APC), choriocarcinoma (CEA), epithelial cell cancer(cyclophilin b), gastric cancer (HER2/neu; c-erbB-2; ga733glycoprotein), hepatocellular cancer (α-fetoprotein), Hodgkins lymphoma(lmp-1; EBNA-1), lung cancer (CEA; MAGE-3; NY-ESO-1), lymphoidcell-derived leukemia (cyclophilin b), melanoma (p15 protein, gp75,oncofetal antigen, GM2 and GD2 gangliosides), myeloma (MUC family;p21ras), non-small cell lung carcinoma (HER2/neu; c-erbB-2),nasopharyngeal cancer (lmp-1; EBNA-1), ovarian cancer (MUC family;HER2/neu; c-erbB-2), prostate cancer (Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3; PSMA; HER2/neu;c-erbB-2), pancreatic cancer (p21ras; MUC family; HER2/neu; c-erbB-2;ga733 glycoprotein), renal cancer (HER2/neu; c-erbB-2), squamous cellcancers of cervix and esophagus (viral products such as human papillomavirus proteins), testicular cancer (NY-ESO-1), T-cell leukemia (HTLV-1epitopes), and melanoma (Melan-A/MART-1; cdc27; MAGE-3; p21ras;gp100^(Pmel117)).

For examples of tumor antigens which bind to either or both MHC class Iand MHC class II molecules, see the following references: Coulie, StemCells 13:393-403, 1995; Traversari et al. J Exp Med 176:1453-1457, 1992;Chaux et al. J Immunol 163:2928-2936, 1999; Fujie et al. Int J Cancer80:169-172, 1999; Tanzarella et al. Cancer Res 59:2668-2674, 1999; vander Bruggen et al. Eur J Immunol 24:2134-2140, 1994; Chaux et al. J ExpMed 189:767-778, 1999; Kawashima et al. Hum Immunol 59:1-14, 1998;Tahara et al. Clin Cancer Res 5:2236-2241, 1999; Gaugler et al. J ExpMed 179:921-930, 1994; van der Bruggen et al. Eur J Immunol24:3038-3043, 1994; Tanaka et al. Cancer Res 57:4465-4468, 1997; Oiso etal. Int J Cancer 81:387-394, 1999; Herman et al. Immunogenetics43:377-383, 1996; Manici et al. J Exp Med 189:871-876, 1999; Duffour etal. Eur J Immunol 29:3329-3337, 1999; Zorn et al. Eur J Immunol29:602-607, 1999; Huang et al. J Immunol 162:6849-6854, 1999; Boël etal. Immunity 2:167-175, 1995; Van den Eynde et al. J Exp Med182:689-698, 1995; De Backer et al. Cancer Res 59:3157-3165, 1999; Jägeret al. J Exp Med 187:265-270, 1998; Wang et al. J Immunol 161:3596-3606,1998; Aarnoudse et al. Int J Cancer 82:442-448, 1999; Guilloux et al. JExp Med 183:1173-1183, 1996; Lupetti et al. J Exp Med 188:1005-1016,1998; Wölfel et al. Eur J Immunol 24:759-764, 1994; Skipper et al. J ExpMed 183:527-534, 1996; Kang et al. J Immunol 155:1343-1348, 1995; Morelet al. Int J Cancer 83:755-759, 1999; Brichard et al. Eur J Immunol26:224-230, 1996; Kittlesen et al. J Immunol 160:2099-2106, 1998;Kawakami et al. J Immunol 161:6985-6992, 1998; Topalian et al. J Exp Med183:1965-1971, 1996; Kobayashi et al. Cancer Research 58:296-301, 1998;Kawakami et al. J Immunol 154:3961-3968, 1995; Tsai et al. J Immunol158:1796-1802, 1997; Cox et al. Science 264:716-719, 1994; Kawakami etal. Proc Natl Acad Sci USA 91:6458-6462, 1994; Skipper et al. J Immunol157:5027-5033, 1996; Robbins et al. J Immunol 159:303-308, 1997;Castelli et al. J Immunol 162:1739-1748, 1999; Kawakami et al. J Exp Med180:347-352, 1994; Castelli et al. J Exp Med 181:363-368, 1995;Schneider et al. Int J Cancer 75:451-458, 1998; Wang et al. J Exp Med183:1131-1140, 1996; Wang et al. J Exp Med 184:2207-2216, 1996;Parkhurst et al. Cancer Research 58:4895-4901, 1998; Tsang et al. J NatlCancer Inst 87:982-990, 1995; Correale et al. J Natl Cancer Inst89:293-300, 1997; Coulie et al. Proc Natl Acad Sci USA 92:7976-7980,1995; Wölfel et al. Science 269:1281-1284, 1995; Robbins et al. J ExpMed 183:1185-1192, 1996; Brändle et al. J Exp Med 183:2501-2508, 1996;ten Bosch et al. Blood 88:3522-3527, 1996; Mandruzzato et al. J Exp Med186:785-793, 1997; Guéguen et al. J Immunol 160:6188-6194, 1998;Gjertsen et al. Int J Cancer 72:784-790, 1997; Gaudin et al. J Immunol162:1730-1738, 1999; Chiari et al. Cancer Res 59:5785-5792, 1999; Hoganet al. Cancer Res 58:5144-5150, 1998; Pieper et al. J Exp Med189:757-765, 1999; Wang et al. Science 284:1351-1354, 1999; Fisk et al.J Exp Med 181:2109-2117, 1995; Brossart et al. Cancer Res 58:732-736,1998; Röpke et al. Proc Natl Acad Sci USA 93:14704-14707, 1996; Ikeda etal. Immunity 6:199-208, 1997; Ronsin et al. J Immunol 163:483-490, 1999;Vonderheide et al. Immunity 10:673-679, 1999. These antigens as well asothers are disclosed in PCT Application PCT/US98/18601

The compositions and methods of the invention can be used alone or inconjunction with other agents and methods useful for the treatment ofcancer. Cancer is currently treated using a variety of modalitiesincluding surgery, radiation therapy and chemotherapy. The choice oftreatment modality will depend upon the type, location and disseminationof the cancer. For example, surgery and radiation therapy may be moreappropriate in the case of solid, well-defined tumor masses and lesspractical in the case of non-solid tumor cancers such as leukemia andlymphoma. One of the advantages of surgery and radiation therapy is theability to control to some extent the impact of the therapy, and thus tolimit the toxicity to normal tissues in the body. However, surgery andradiation therapy are often followed by chemotherapy to guard againstany remaining or radio-resistant cancer cells. Chemotherapy is also themost appropriate treatment for disseminated cancers such as leukemia andlymphoma as well as metastases.

Chemotherapy refers to therapy using chemical and/or biological agentsto attack cancer cells. Unlike localized surgery or radiation,chemotherapy is generally administered in a systemic fashion and thustoxicity to normal tissues is a major concern. Because many chemotherapyagents target cancer cells based on their proliferative profiles,tissues such as the gastrointestinal tract and the bone marrow which arenormally proliferative are also susceptible to the effects of thechemotherapy. One of the major side effects of chemotherapy ismyelosuppression (including anemia, neutropenia and thrombocytopenia)which results from the death of normal hemopoietic precursors.

Many chemotherapeutic agents have been developed for the treatment ofcancer. Not all tumors, however, respond to chemotherapeutic agents andothers although initially responsive to chemotherapeutic agents maydevelop resistance. As a result, the search for effective anti-cancerdrugs has intensified in an effort to find even more effective agentswith less non-specific toxicity.

Cancer medicaments function in a variety of ways. Some cancermedicaments work by targeting physiological mechanisms that are specificto tumor cells. Examples include the targeting of specific genes andtheir gene products (i.e., proteins primarily) which are mutated incancers. Such genes include but are not limited to oncogenes (e.g., Ras,Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cellcycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments canalternately target signal transduction pathways and molecular mechanismswhich are altered in cancer cells. Targeting of cancer cells via theepitopes expressed on their cell surface is accomplished through the useof monoclonal antibodies. This latter type of cancer medicament isgenerally referred to herein as immunotherapy.

Other cancer medicaments target cells other than cancer cells. Forexample, some medicaments prime the immune system to attack tumor cells(i.e., cancer vaccines). Still other medicaments, called angiogenesisinhibitors, function by attacking the blood supply of solid tumors.Since the most malignant cancers are able to metastasize (i.e., exit theprimary tumor site and seed a another site, thereby forming a secondarytumor), medicaments that impede this metastasis are also useful in thetreatment of cancer. Angiogenic mediators include basic FGF, VEGF,angiopoietins, angiostatin, endostatin, TNF-α, TNP-470,thrombospondin-1, platelet factor 4, CAI, and certain members of theintegrin family of proteins. One category of this type of medicament isa metalloproteinase inhibitor, which inhibits the enzymes used by thecancer cells to exist the primary tumor site and extravasate intoanother tissue.

Some cancer cells are antigenic and thus can be targeted by the immunesystem. In one aspect, the combined administration of nucleic acid andcancer medicaments, particularly those which are classified as cancerimmunotherapies, is useful for stimulating a specific immune responseagainst a cancer antigen.

The theory of immune surveillance is that a prime function of the immunesystem is to detect and eliminate neoplastic cells before a tumor forms.A basic principle of this theory is that cancer cells are antigenicallydifferent from normal cells and thus elicit immune reactions that aresimilar to those that cause rejection of immunologically incompatibleallografts. Studies have confirmed that tumor cells differ, eitherqualitatively or quantitatively, in their expression of antigens. Forexample, “tumor-specific antigens” are antigens that are specificallyassociated with tumor cells but not normal cells. Examples of tumorspecific antigens are viral antigens in tumors induced by DNA or RNAviruses. “Tumor-associated” antigens are present in both tumor cells andnormal cells but are present in a different quantity or a different formin tumor cells. Examples of such antigens are oncofetal antigens (e.g.,carcinoembryonic antigen), differentiation antigens (e.g., T and Tnantigens), and oncogene products (e.g., HER/neu).

Different types of cells that can kill tumor targets in vitro and invivo have been identified: natural killer (NK) cells, cytolytic Tlymphocytes (CTLs), lymphokine-activated killer cells (LAKs), andactivated macrophages. NK cells can kill tumor cells without having beenpreviously sensitized to specific antigens, and the activity does notrequire the presence of class I antigens encoded by the majorhistocompatibility complex (MHC) on target cells. NK cells are thoughtto participate in the control of nascent tumors and in the control ofmetastatic growth. In contrast to NK cells, CTLs can kill tumor cellsonly after they have been sensitized to tumor antigens and when thetarget antigen is expressed on the tumor cells that also express MHCclass I. CTLs are thought to be effector cells in the rejection oftransplanted tumors and of tumors caused by DNA viruses. LAK cells are asubset of null lymphocytes distinct from the NK and CTL populations.Activated macrophages can kill tumor cells in a manner that is neitherantigen-dependent nor MHC-restricted once activated. Activatedmacrophages are through to decrease the growth rate of the tumors theyinfiltrate. In vitro assays have identified other immune mechanisms suchas antibody-dependent, cell-mediated cytotoxic reactions and lysis byantibody plus complement. However, these immune effector mechanisms arethought to be less important in vivo than the function of NK, CTLs, LAK,and macrophages in vivo (for review see Piessens W F et al. “TumorImmunology”, In: Scientific American Medicine, Vol. 2, ScientificAmerican Books, N.Y., pp. 1-13, 1996).

The goal of immunotherapy is to augment a patient's immune response toan established tumor. One method of immunotherapy includes the use ofadjuvants. Adjuvant substances derived from microorganisms, such asbacillus Calmette-Guérin, heighten the immune response and enhanceresistance to tumors in animals.

Immunotherapeutic agents are medicaments which derive from antibodies orantibody fragments which specifically bind or recognize a cancerantigen. Antibody-based immunotherapies may function by binding to thecell surface of a cancer cell and thereby stimulate the endogenousimmune system to attack the cancer cell. Another way in whichantibody-based therapy functions is as a delivery system for thespecific targeting of toxic substances to cancer cells. Antibodies areusually conjugated to toxins such as ricin (e.g., from castor beans),calicheamicin and maytansinoids, to radioactive isotopes such asIodine-131 and Yttrium-90, to chemotherapeutic agents (as describedherein), or to biological response modifiers. In this way, the toxicsubstances can be concentrated in the region of the cancer andnon-specific toxicity to normal cells can be minimized. In addition tothe use of antibodies which are specific for cancer antigens, antibodieswhich bind to vasculature, such as those which bind to endothelialcells, are also useful in the invention. This is because solid tumorsgenerally are dependent upon newly formed blood vessels to survive, andthus most tumors are capable of recruiting and stimulating the growth ofnew blood vessels. As a result, one strategy of many cancer medicamentsis to attack the blood vessels feeding a tumor and/or the connectivetissues (or stroma) supporting such blood vessels.

Cancer vaccines are medicaments which are intended to stimulate anendogenous immune response against cancer cells. Currently producedvaccines predominantly activate the humoral immune system (i.e., theantibody-dependent immune response). Other vaccines currently indevelopment are focused on activating the cell-mediated immune systemincluding cytotoxic T lymphocytes which are capable of killing tumorcells. Cancer vaccines generally enhance the presentation of cancerantigens to both antigen presenting cells (e.g., macrophages anddendritic cells) and/or to other immune cells such as T cells, B cells,and NK cells.

Although cancer vaccines may take one of several forms, as discussedinfra, their purpose is to deliver cancer antigens and/or cancerassociated antigens to antigen presenting cells (APC) in order tofacilitate the endogenous processing of such antigens by APC and theultimate presentation of antigen presentation on the cell surface in thecontext of MHC class I molecules. One form of cancer vaccine is a wholecell vaccine which is a preparation of cancer cells which have beenremoved from a subject, treated ex vivo and then reintroduced as wholecells in the subject. Lysates of tumor cells can also be used as cancervaccines to elicit an immune response. Another form cancer vaccine is apeptide vaccine which uses cancer-specific or cancer-associated smallproteins to activate T cells. Cancer-associated proteins are proteinswhich are not exclusively expressed by cancer cells (i.e., other normalcells may still express these antigens). However, the expression ofcancer-associated antigens is generally consistently upregulated withcancers of a particular type. Other cancer vaccines include gangliosidevaccines, heat-shock protein vaccines, viral and bacterial vaccines, andnucleic acid vaccines.

Yet another form of cancer vaccine is a dendritic cell vaccine whichincludes whole dendritic cells which have been exposed to a cancerantigen or a cancer-associated antigen in vitro. Lysates or membranefractions of dendritic cells may also be used as cancer vaccines.Dendritic cell vaccines are able to activate APCs directly. A dendriticcell is a professional APC. Dendritic cells form the link between theinnate and the acquired immune system by presenting antigens and throughtheir expression of pattern recognition receptors which detect microbialmolecules like LPS in their local environment. Dendritic cellsefficiently internalize, process, and present soluble specific antigento which it is exposed. The process of internalizing and presentingantigen causes rapid upregulation of the expression of majorhistocompatibility complex (MHC) and costimulatory molecules, theproduction of cytokines, and migration toward lymphatic organs wherethey are believed to be involved in the activation of T cells.

As used herein, chemotherapeutic agents embrace all other forms ofcancer medicaments which do not fall into the categories ofimmunotherapeutic agents or cancer vaccines. Chemotherapeutic agents asused herein encompass both chemical and biological agents. These agentsfunction to inhibit a cellular activity which the cancer cell isdependent upon for continued survival. Categories of chemotherapeuticagents include alkylating/alkaloid agents, antimetabolites, hormones orhormone analogs, and miscellaneous antineoplastic drugs. Most if not allof these agents are directly toxic to cancer cells and do not requireimmune stimulation.

An “infectious disease” or, equivalently, an “infection” as used herein,refers to a disorder arising from the invasion of a host, superficially,locally, or systemically, by an infectious organism. Infectiousorganisms include bacteria, viruses, fungi, and parasites. Accordingly,“infectious disease” includes bacterial infections, viral infections,fungal infections and parasitic infections.

A subject having an infectious disease is a subject that has beenexposed to an infectious organism and has acute or chronic detectablelevels of the organism in the body. Exposure to the infectious organismgenerally occurs with the external surface of the subject, e.g., skin ormucosal membranes and/or refers to the penetration of the externalsurface of the subject by the infectious organism.

A subject at risk of developing an infectious disease is a subject whohas a higher than normal risk of exposure to an infection causingpathogen. For instance, a subject at risk may be a subject who isplanning to travel to an area where a particular type of infectiousagent is found or it may be a subject who through lifestyle or medicalprocedures is exposed to bodily fluids which may contain infectiousorganisms or directly to the organism or a subject living in an areawhere an infectious organism has been identified. Subjects at risk ofdeveloping an infectious disease also include general populations towhich a medical agency recommends vaccination against a particularinfectious organism.

A subject at risk of developing an infectious disease includes thosesubjects that have a general risk of exposure to a microorganism, e.g.,influenza, but that do not have the active disease during the treatmentof the invention, as well as subjects that are considered to be atspecific risk of developing an infectious disease because of medical orenvironmental factors that expose the subject to a particularmicroorganism.

Bacteria are unicellular organisms which multiply asexually by binaryfission. They are classified and named based on their morphology,staining reactions, nutrition and metabolic requirements, antigenicstructure, chemical composition, and genetic homology. Bacteria can beclassified into three groups based on their morphological forms,spherical (coccus), straight-rod (bacillus) and curved or spiral rod(vibrio, campylobacter, spirillum, and spirochaete). Bacteria are alsomore commonly characterized based on their staining reactions into twoclasses of organisms, gram-positive and gram-negative. Gram refers tothe method of staining which is commonly performed in microbiology labs.Gram-positive organisms retain the stain following the stainingprocedure and appear a deep violet color. Gram-negative organisms do notretain the stain but take up the counter-stain and thus appear pink.

Infectious bacteria include, but are not limited to, gram negative andgram positive bacteria. Gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansasii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic species), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus anthracis, Corynebacterium diphtheriae,Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Viruses are small infectious agents which generally contain a nucleicacid core and a protein coat, but are not independently livingorganisms. Viruses can also take the form of infectious nucleic acidslacking a protein. A virus cannot survive in the absence of a livingcell within which it can replicate. Viruses enter specific living cellseither by endocytosis or direct injection of DNA (phage) and multiply,causing disease. The multiplied virus can then be released and infectadditional cells. Some viruses are DNA-containing viruses and others areRNA-containing viruses. In some aspects, the invention also intends totreat diseases in which prions are implicated in disease progressionsuch as for example bovine spongiform encephalopathy (i.e., mad cowdisease, BSE) or scrapie infection in animals, or Creutzfeldt-Jakobdisease in humans.

Viruses include, but are not limited to, enteroviruses (including, butnot limited to, viruses that the family picornaviridae, such as poliovirus, coxsackie virus, echo virus), rotaviruses, adenovirus, hepatitisvirus. Specific examples of viruses that have been found in humansinclude but are not limited to: Retroviridae (e.g., humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses);Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papillomaviruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV));Poxviridae (variola viruses, vaccinia viruses, pox viruses);Iridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the etiological agents of spongiform encephalopathies, the agentof delta hepatitis (thought to be a defective satellite of hepatitis Bvirus), the agents of non-A, non-B hepatitis (class 1=internallytransmitted; class 2=parenterally transmitted (i.e., Hepatitis C);Norwalk and related viruses, and astroviruses).

Fungi are eukaryotic organisms, only a few of which cause infection invertebrate mammals. Because fungi are eukaryotic organisms, they differsignificantly from prokaryotic bacteria in size, structuralorganization, life cycle and mechanism of multiplication. Fungi areclassified generally based on morphological features, modes ofreproduction and culture characteristics. Although fungi can causedifferent types of disease in subjects, such as respiratory allergiesfollowing inhalation of fungal antigens, fungal intoxication due toingestion of toxic substances, such as Amanita phalloides toxin andphallotoxin produced by poisonous mushrooms and aflatoxins, produced byaspergillus species, not all fungi cause infectious disease.

Infectious fungi can cause systemic or superficial infections. Primarysystemic infection can occur in normal healthy subjects, andopportunistic infections are most frequently found in immunocompromisedsubjects. The most common fungal agents causing primary systemicinfection include Blastomyces, Coccidioides, and Htoplasma. Common fungicausing opportunistic infection in immunocompromised or immunosuppressedsubjects include, but are not limited to, Candida albicans, Cryptococcusneoformans, and various Aspergillus species. Systemic fungal infectionsare invasive infections of the internal organs. The organism usuallyenters the body through the lungs, gastrointestinal tract, orintravenous catheters. These types of infections can be caused byprimary pathogenic fungi or opportunistic fungi.

Superficial fungal infections involve growth of fungi on an externalsurface without invasion of internal tissues. Typical superficial fungalinfections include cutaneous fungal infections involving skin, hair, ornails.

Diseases associated with fungal infection include aspergillosis,blastomycosis, candidiasis, chromoblastomycosis, coccidioidomycosis,cryptococcosis, fungal eye infections, fungal hair, nail, and skininfections, histoplasmosis, lobomycosis, mycetoma, otomycosis,paracoccidioidomycosis, disseminated Penicillium marneffei,phaeohyphomycosis, rhinosporidioisis, sporotrichosis, and zygomycosis.

Parasites are organisms which depend upon other organisms in order tosurvive and thus must enter, or infect, another organism to continuetheir life cycle. The infected organism, i.e., the host, provides bothnutrition and habitat to the parasite. Although in its broadest sensethe term parasite can include all infectious agents (i.e., bacteria,viruses, fungi, protozoa and helminths), generally speaking, the term isused to refer solely to protozoa, helminths, and ectoparasiticarthropods (e.g., ticks, mites, etc.). Protozoa are single-celledorganisms which can replicate both intracellularly and extracellularly,particularly in the blood, intestinal tract or the extracellular matrixof tissues. Helminths are multicellular organisms which almost alwaysare extracellular (an exception being Trichinella spp.). Helminthsnormally require exit from a primary host and transmission into asecondary host in order to replicate. In contrast to theseaforementioned classes, ectoparasitic arthropods form a parasiticrelationship with the external surface of the host body.

Parasites include intracellular parasites and obligate intracellularparasites. Examples of parasites include but are not limited toPlasmodium falciparum, Plasmodium ovale, Plasmodium malariae,Plasmdodium vivax, Plasmodium knowlesi, Babesia microti, Babesiadivergens, Trypanosoma cruzi, Toxoplasma gondii, Trichinella spiralis,Leishmania major, Leishmania donovani, Leishmania braziliensis,Leishmania tropica, Trypanosoma gambiense, Trypanosoma rhodesiense andSchistosoma mansoni.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference. Each of the foregoing lists isillustrative and is not intended to be limiting.

The compositions and methods of the invention can be used alone or inconjunction with other agents and methods useful for the treatment ofinfection. Infection medicaments include but are not limited toanti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“antibiotic”, “anti-bacterial agent”, “anti-viral agent”, “anti-fungalagent”, “anti-parasitic agent” and “parasiticide” have well-establishedmeanings to those of ordinary skill in the art and are defined instandard medical texts. Briefly, anti-bacterial agents kill or inhibitbacteria, and include antibiotics as well as other synthetic or naturalcompounds having similar functions. Anti-viral agents can be isolatedfrom natural sources or synthesized and are useful for killing orinhibiting viruses. Anti-fungal agents are used to treat superficialfungal infections as well as opportunistic and primary systemic fungalinfections. Anti-parasite agents kill or inhibit parasites. Manyantibiotics are low molecular weight molecules which are produced assecondary metabolites by cells, such as microorganisms. In general,antibiotics interfere with one or more functions or structures which arespecific for the microorganism and which are not present in host cells.

One of the problems with anti-infective therapies is the side effectsoccurring in the host that is treated with the anti-infective agent. Forinstance, many anti-infectious agents can kill or inhibit a broadspectrum of microorganisms and are not specific for a particular type ofspecies. Treatment with these types of anti-infectious agents results inthe killing of the normal microbial flora living in the host, as well asthe infectious microorganism. The loss of the microbial flora can leadto disease complications and predispose the host to infection by otherpathogens, since the microbial flora compete with and function asbarriers to infectious pathogens. Other side effects may arise as aresult of specific or non-specific effects of these chemical entities onnon-microbial cells or tissues of the host.

Another problem with widespread use of anti-infectants is thedevelopment of antibiotic-resistant strains of microorganisms. Already,vancomycin-resistant enterococci, penicillin-resistant pneumococci,multi-resistant S. aureus, and multi-resistant tuberculosis strains havedeveloped and are becoming major clinical problems. Widespread use ofanti-infectants will likely produce many antibiotic-resistant strains ofbacteria. As a result, new anti-infective strategies will be required tocombat these microorganisms.

Antibacterial antibiotics which are effective for killing or inhibitinga wide range of bacteria are referred to as broad-spectrum antibiotics.Other types of antibacterial antibiotics are predominantly effectiveagainst the bacteria of the class gram-positive or gram-negative. Thesetypes of antibiotics are referred to as narrow-spectrum antibiotics.Other antibiotics which are effective against a single organism ordisease and not against other types of bacteria, are referred to aslimited-spectrum antibiotics.

Anti-bacterial agents are sometimes classified based on their primarymode of action. In general, anti-bacterial agents are cell wallsynthesis inhibitors, cell membrane inhibitors, protein synthesisinhibitors, nucleic acid synthesis or functional inhibitors, andcompetitive inhibitors. Cell wall synthesis inhibitors inhibit a step inthe process of cell wall synthesis, and in general in the synthesis ofbacterial peptidoglycan. Cell wall synthesis inhibitors include β-lactamantibiotics, natural penicillins, semi-synthetic penicillins,ampicillin, clavulanic acid, cephalolsporins, and bacitracin.

The β-lactams are antibiotics containing a four-membered β-lactam ringwhich inhibits the last step of peptidoglycan synthesis. β-lactamantibiotics can be synthesized or natural. The β-lactam antibioticsproduced by penicillium are the natural penicillins, such as penicillinG or penicillin V. These are produced by fermentation of Penicilliumchrysogenum. The natural penicillins have a narrow spectrum of activityand are generally effective against Streptococcus, Gonococcus, andStaphylococcus. Other types of natural penicillins, which are alsoeffective against gram-positive bacteria, include penicillins F, X, K,and O.

Semi-synthetic penicillins are generally modifications of the molecule6-aminopenicillanic acid produced by a mold. The 6-aminopenicillanicacid can be modified by addition of side chains which producepenicillins having broader spectrums of activity than naturalpenicillins or various other advantageous properties. Some types ofsemi-synthetic penicillins have broad spectrums against gram-positiveand gram-negative bacteria, but are inactivated by penicillinase. Thesesemi-synthetic penicillins include ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, and piperacillin. Other types of semi-syntheticpenicillins have narrower activities against gram-positive bacteria, buthave developed properties such that they are not inactivated bypenicillinase. These include, for instance, methicillin, dicloxacillin,and nafcillin. Some of the broad spectrum semi-synthetic penicillins canbe used in combination with β-lactamase inhibitors, such as clavulanicacids and sulbactam. The β-lactamase inhibitors do not haveanti-microbial action but they function to inhibit penicillinase, thusprotecting the semi-synthetic penicillin from degradation.

One of the serious side effects associated with penicillins, bothnatural and semi-synthetic, is penicillin allergy. Penicillin allergiesare very serious and can cause death rapidly. In a subject that isallergic to penicillin, the β-lactam molecule will attach to a serumprotein which initiates an IgE-mediated inflammatory response. Theinflammatory response leads to anaphylaxis and possibly death.

Another type of β-lactam antibiotic is the cephalolsporins. They aresensitive to degradation by bacterial β-lactamases, and thus, are notalways effective alone. Cephalolsporins, however, are resistant topenicillinase. They are effective against a variety of gram-positive andgram-negative bacteria. Cephalolsporins include, but are not limited to,cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin,cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime,ceftriaxone, cefoperazone, ceftazidine, and moxalactam.

Bacitracin is another class of antibiotics which inhibit cell wallsynthesis, by inhibiting the release of muropeptide subunits orpeptidoglycan from the molecule that delivers the subunit to the outsideof the membrane. Although bacitracin is effective against gram-positivebacteria, its use is limited in general to topical administrationbecause of its high toxicity.

Carbapenems are another broad-spectrum β-lactam antibiotic, which iscapable of inhibiting cell wall synthesis. Examples of carbapenemsinclude, but are not limited to, imipenems. Monobactams are alsobroad-spectrum β-lactam antibiotics, and include, euztreonam. Anantibiotic produced by Streptomyces, vancomycin, is also effectiveagainst gram-positive bacteria by inhibiting cell membrane synthesis.

Another class of anti-bacterial agents is the anti-bacterial agents thatare cell membrane inhibitors. These compounds disorganize the structureor inhibit the function of bacterial membranes. One problem withanti-bacterial agents that are cell membrane inhibitors is that they canproduce effects in eukaryotic cells as well as bacteria because of thesimilarities in phospholipids in bacterial and eukaryotic membranes.Thus these compounds are rarely specific enough to permit thesecompounds to be used systemically and prevent the use of high doses forlocal administration.

One clinically useful cell membrane inhibitor is Polymyxin. Polymyxinsinterfere with membrane function by binding to membrane phospholipids.Polymyxin is effective mainly against Gram-negative bacteria and isgenerally used in severe Pseudomonas infections or Pseudomonasinfections that are resistant to less toxic antibiotics. The severe sideeffects associated with systemic administration of this compound includedamage to the kidney and other organs.

Other cell membrane inhibitors include Amphotericin B and Nystatin whichare anti-fungal agents used predominantly in the treatment of systemicfungal infections and Candida yeast infections. Imidazoles are anotherclass of antibiotic that is a cell membrane inhibitor. Imidazoles areused as anti-bacterial agents as well as anti-fungal agents, e.g., usedfor treatment of yeast infections, dermatophytic infections, andsystemic fungal infections. Imidazoles include but are not limited toclotrimazole, miconazole, ketoconazole, itraconazole, and fluconazole.

Many anti-bacterial agents are protein synthesis inhibitors. Thesecompounds prevent bacteria from synthesizing structural proteins andenzymes and thus cause inhibition of bacterial cell growth or functionor cell death. In general these compounds interfere with the processesof transcription or translation. Anti-bacterial agents that blocktranscription include but are not limited to Rifampins and Ethambutol.Rifampins, which inhibit the enzyme RNA polymerase, have a broadspectrum activity and are effective against gram-positive andgram-negative bacteria as well as Mycobacterium tuberculosis. Ethambutolis effective against Mycobacterium tuberculosis.

Anti-bacterial agents which block translation interfere with bacterialribosomes to prevent mRNA from being translated into proteins. Ingeneral this class of compounds includes but is not limited totetracyclines, chloramphenicol, the macrolides (e.g., erythromycin) andthe aminoglycosides (e.g., streptomycin).

The aminoglycosides are a class of antibiotics which are produced by thebacterium Streptomyces, such as, for instance streptomycin, kanamycin,tobramycin, amikacin, and gentamicin. Aminoglycosides have been usedagainst a wide variety of bacterial infections caused by Gram-positiveand Gram-negative bacteria. Streptomycin has been used extensively as aprimary drug in the treatment of tuberculosis. Gentamicin is usedagainst many strains of Gram-positive and Gram-negative bacteria,including Pseudomonas infections, especially in combination withTobramycin. Kanamycin is used against many Gram-positive bacteria,including penicillin-resistant Staphylococci. One side effect ofaminoglycosides that has limited their use clinically is that at dosageswhich are essential for efficacy, prolonged use has been shown to impairkidney function and cause damage to the auditory nerves leading todeafness.

Another type of translation inhibitor anti-bacterial agent is thetetracyclines. The tetracyclines are a class of antibiotics that arebroad-spectrum and are effective against a variety of gram-positive andgram-negative bacteria. Examples of tetracyclines include tetracycline,minocycline, doxycycline, and chlortetracycline. They are important forthe treatment of many types of bacteria but are particularly importantin the treatment of Lyme disease. As a result of their low toxicity andminimal direct side effects, the tetracyclines have been overused andmisused by the medical community, leading to problems. For instance,their overuse has led to widespread development of resistance.

Anti-bacterial agents such as the macrolides bind reversibly to the 50 Sribosomal subunit and inhibit elongation of the protein by peptidyltransferase or prevent the release of uncharged tRNA from the bacterialribosome or both. These compounds include erythromycin, roxithromycin,clarithromycin, oleandomycin, and azithromycin. Erythromycin is activeagainst most Gram-positive bacteria, Neisseria, Legionella andHaemophilus, but not against the Enterobacteriaceae. Lincomycin andclindamycin, which block peptide bond formation during proteinsynthesis, are used against gram-positive bacteria.

Another type of translation inhibitor is chloramphenicol.Chloramphenicol binds the 70 S ribosome inhibiting the bacterial enzymepeptidyl transferase thereby preventing the growth of the polypeptidechain during protein synthesis. One serious side effect associated withchloramphenicol is aplastic anemia. Aplastic anemia develops at doses ofchloramphenicol which are effective for treating bacteria in a smallproportion (1/50,000) of patients. Chloramphenicol which was once ahighly prescribed antibiotic is now seldom uses as a result of thedeaths from anemia. Because of its effectiveness it is still used inlife-threatening situations (e.g., typhoid fever).

Some anti-bacterial agents disrupt nucleic acid synthesis or function,e.g., bind to DNA or RNA so that their messages cannot be read. Theseinclude but are not limited to quinolones and co-trimoxazole, bothsynthetic chemicals and rifamycins, a natural or semi-syntheticchemical. The quinolones block bacterial DNA replication by inhibitingthe DNA gyrase, the enzyme needed by bacteria to produce their circularDNA. They are broad spectrum and examples include norfloxacin,ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acidis a bactericidal agent that binds to the DNA gyrase enzyme(topoisomerase) which is essential for DNA replication and allowssupercoils to be relaxed and reformed, inhibiting DNA gyrase activity.The main use of nalidixic acid is in treatment of lower urinary tractinfections (UTI) because it is effective against several types ofGram-negative bacteria such as E. coli, Enterobacter aerogenes, K.pneumoniae and Proteus species which are common causes of UTI.Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim,which blocks the bacterial synthesis of folic acid needed to make DNAnucleotides. Rifampicin is a derivative of rifamycin that is activeagainst Gram-positive bacteria (including Mycobacterium tuberculosis andmeningitis caused by Neisseria meningitidis) and some Gram-negativebacteria. Rifampicin binds to the beta subunit of the polymerase andblocks the addition of the first nucleotide which is necessary toactivate the polymerase, thereby blocking mRNA synthesis.

Another class of anti-bacterial agents is compounds that function ascompetitive inhibitors of bacterial enzymes. The competitive inhibitorsare mostly all structurally similar to a bacterial growth factor andcompete for binding but do not perform the metabolic function in thecell. These compounds include sulfonamides and chemically modified formsof sulfanilamide which have even higher and broader antibacterialactivity. The sulfonamides (e.g., gantrisin and trimethoprim) are usefulfor the treatment of Streptococcus pneumoniae, beta-hemolyticstreptococci and E. coli, and have been used in the treatment ofuncomplicated UTI caused by E. coli, and in the treatment ofmeningococcal meningitis.

Anti-viral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleoside analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Another category of anti-viral agents are nucleoside analogues.Nucleoside analogues are synthetic compounds which are similar tonucleosides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleoside analogues are in the cell, they arephosphorylated, producing the triphosphate form which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleoside analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleoside analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine).

Another class of anti-viral agents includes cytokines such asinterferons. The interferons are cytokines which are secreted byvirus-infected cells as well as immune cells. The interferons functionby binding to specific receptors on cells adjacent to the infectedcells, causing the change in the cell which protects it from infectionby the virus. α and β-interferon also induce the expression of Class Iand Class II MHC molecules on the surface of infected cells, resultingin increased antigen presentation for host immune cell recognition. αand β-interferons are available as recombinant forms and have been usedfor the treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Immunoglobulin therapy is used for the prevention of viral infection.Immunoglobulin therapy for viral infections is different from bacterialinfections, because rather than being antigen-specific, theimmunoglobulin therapy functions by binding to extracellular virions andpreventing them from attaching to and entering cells which aresusceptible to the viral infection. The therapy is useful for theprevention of viral infection for the period of time that the antibodiesare present in the host. In general there are two types ofimmunoglobulin therapies, normal immune globulin therapy andhyper-immune globulin therapy. Normal immune globulin therapy utilizes aantibody product which is prepared from the serum of normal blood donorsand pooled. This pooled product contains low titers of antibody to awide range of human viruses, such as hepatitis A, parvovirus,enterovirus (especially in neonates). Hyper-immune globulin therapyutilizes antibodies which are prepared from the serum of individuals whohave high titers of an antibody to a particular virus. Those antibodiesare then used against a specific virus. Examples of hyper-immuneglobulins include zoster immune globulin (useful for the prevention ofvaricella in immunocompromised children and neonates), human rabiesimmune globulin (useful in the post-exposure prophylaxis of a subjectbitten by a rabid animal), hepatitis B immune globulin (useful in theprevention of hepatitis B virus, especially in a subject exposed to thevirus), and RSV immune globulin (useful in the treatment of respiratorysyncitial virus infections).

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,imidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g., chitinase) or immunosuppression (501 cream).

Parasiticides are agents that kill parasites directly. Such compoundsare known in the art and are generally commercially available. Examplesof parasiticides useful for human administration include but are notlimited to albendazole, amphotericin B, benznidazole, bithionol,chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine,diethylcarbamazine, diloxamide furoate, eflornithine, furazolidaone,glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole,mefloquine, meglumine antimoniate, melarsoprol, metrifonate,metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin,pentamidine isethionate, piperazine, praziquantel, primaquine phosphate,proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides,pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidinegluconate, spiramycin, stibogluconate sodium (sodium antimonygluconate), suramin, tetracycline, doxycycline, thiabendazole,tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide.

The compositions and methods of the invention may also find use in thetreatment of allergy and asthma.

An “allergy” refers to acquired hypersensitivity to a substance(allergen). Allergic conditions include but are not limited to eczema,allergic rhinitis or coryza, hay fever, allergic conjunctivitis,bronchial asthma, urticaria (hives) and food allergies, other atopicconditions including atopic dermatitis; anaphylaxis; drug allergy; andangioedema. Allergic diseases include but are not limited to rhinitis(hay fever), asthma, urticaria, and atopic dermatitis.

Allergy is a disease associated with the production of antibodies from aparticular class of immunoglobulin, IgE, against allergens. Thedevelopment of an IgE-mediated response to common aeroallergens is alsoa factor which indicates predisposition towards the development ofasthma. If an allergen encounters a specific IgE which is bound to anIgE Fc receptor (FcεR) on the surface of a basophil (circulating in theblood) or mast cell (dispersed throughout solid tissue), the cellbecomes activated, resulting in the production and release of mediatorssuch as histamine, serotonin, and lipid mediators.

A subject having an allergy is a subject that is currently experiencingor has previously experienced an allergic reaction in response to anallergen.

A subject at risk of developing an allergy or asthma is a subject thathas been identified as having an allergy or asthma in the past but whois not currently experiencing the active disease, as well as a subjectthat is considered to be at risk of developing asthma or allergy becauseof genetic or environmental factors. A subject at risk of developingallergy or asthma can also include a subject who has any risk ofexposure to an allergen or a risk of developing asthma, i.e., someonewho has suffered from an asthmatic attack previously or has apredisposition to asthmatic attacks. For instance, a subject at risk maybe a subject who is planning to travel to an area where a particulartype of allergen or asthmatic initiator is found or it may even be anysubject living in an area where an allergen has been identified. If thesubject develops allergic responses to a particular antigen and thesubject may be exposed to the antigen, i.e., during pollen season, thenthat subject is at risk of exposure to the antigen.

The generic name for molecules that cause an allergic reaction isallergen. An “allergen” as used herein is a molecule capable ofprovoking an immune response characterized by production of IgE. Anallergen is a substance that can induce an allergic or asthmaticresponse in a susceptible subject. Thus, in the context of thisinvention, the term allergen means a specific type of antigen which cantrigger an allergic response which is mediated by IgE antibody. Themethod and preparations of this invention extend to a broad class ofsuch allergens and fragments of allergens or haptens acting asallergens. The list of allergens is enormous and can include pollens,insect venoms, animal dander, dust, fungal spores, and drugs (e.g.,penicillin).

There are numerous species of allergens. The allergic reaction occurswhen tissue-sensitizing immunoglobulin of the IgE type reacts withforeign allergen. The IgE antibody is bound to mast cells and/orbasophils, and these specialized cells release chemical mediators(vasoactive amines) of the allergic reaction when stimulated to do so byallergens bridging the ends of the antibody molecule. Htamine, plateletactivating factor, arachidonic acid metabolites, and serotonin are amongthe best known mediators of allergic reactions in man. Htamine and theother vasoactive amines are normally stored in mast cells and basophilleukocytes. The mast cells are dispersed throughout animal tissue andthe basophils circulate within the vascular system. These cellsmanufacture and store histamine within the cell unless the specializedsequence of events involving IgE binding occurs to trigger its release.

The symptoms of the allergic reaction vary, depending on the locationwithin the body where the IgE reacts with the antigen. If the reactionoccurs along the respiratory epithelium, the symptoms are sneezing,coughing and asthmatic reactions. If the interaction occurs in thedigestive tract, as in the case of food allergies, abdominal pain anddiarrhea are common. Systemic reactions, for example following a beesting, can be severe and often life-threatening.

Delayed-type hypersensitivity, also known as type IV allergy reaction,is an allergic reaction characterized by a delay period of at least 12hours from invasion of the antigen into the allergic subject untilappearance of the inflammatory or immune reaction. The T lymphocytes(sensitized T lymphocytes) of individuals in an allergic condition reactwith the antigen, triggering the T lymphocytes to release lymphokines(macrophage migration inhibitory factor (MIF), macrophage activatingfactor (MAF), mitogenic factor (MF), skin-reactive factor (SRF),chemotactic factor, neovascularization-accelerating factor, etc.), whichfunction as inflammation mediators, and the biological activity of theselymphokines, together with the direct and indirect effects of locallyappearing lymphocytes and other inflammatory immune cells, give rise tothe type IV allergy reaction. Delayed allergy reactions includetuberculin type reaction, homograft rejection reaction, cell-dependenttype protective reaction, contact dermatitis hypersensitivity reaction,and the like, which are known to be most strongly suppressed bysteroidal agents. Consequently, steroidal agents are effective againstdiseases which are caused by delayed allergy reactions. Long-term use ofsteroidal agents at concentrations currently being used can, however,lead to the serious side-effect known as steroid dependence. The methodsof the invention solve some of these problems, by providing for lowerand fewer doses to be administered.

Immediate hypersensitivity (or anaphylactic response) is a form ofallergic reaction which develops very quickly, i.e., within seconds orminutes of exposure of the patient to the causative allergen, and it ismediated by IgE antibodies made by B lymphocytes. In nonallergicpatients, there is no IgE antibody of clinical relevance; but, in aperson suffering with allergic diseases, IgE antibody mediates immediatehypersensitivity by sensitizing mast cells which are abundant in theskin, lymphoid organs, in the membranes of the eye, nose and mouth, andin the respiratory tract and intestines.

Mast cells have surface receptors for IgE, and the IgE antibodies inallergy-suffering patients become bound to them. As discussed brieflyabove, when the bound IgE is subsequently contacted by the appropriateallergen, the mast cell is caused to degranulate and to release varioussubstances called bioactive mediators, such as histamine, into thesurrounding tissue. It is the biologic activity of these substanceswhich is responsible for the clinical symptoms typical of immediatehypersensitivity; namely, contraction of smooth muscle in the airways orthe intestine, the dilation of small blood vessels and the increase intheir permeability to water and plasma proteins, the secretion of thicksticky mucus, and in the skin, redness, swelling and the stimulation ofnerve endings that results in itching or pain.

“Asthma” as used herein refers to a disorder of the respiratory systemcharacterized by inflammation, narrowing of the airways, and increasedreactivity of the airways to inhaled agents. Asthma is frequently,although not exclusively, associated with an atopic or allergiccondition. Symptoms of asthma include recurrent episodes of wheezing,breathlessness, and chest tightness, and coughing, resulting fromairflow obstruction. Airway inflammation associated with asthma can bedetected through observation of a number of physiological changes, suchas, denudation of airway epithelium, collagen deposition beneathbasement membrane, edema, mast cell activation, inflammatory cellinfiltration, including neutrophils, inosineophils, and lymphocytes. Asa result of the airway inflammation, asthma patients often experienceairway hyper-responsiveness, airflow limitation, respiratory symptoms,and disease chronicity. Airflow limitations include acutebronchoconstriction, airway edema, mucous plug formation, and airwayremodeling, features which often lead to bronchial obstruction. In somecases of asthma, sub-basement membrane fibrosis may occur, leading topersistent abnormalities in lung function.

Research over the past several years has revealed that asthma likelyresults from complex interactions among inflammatory cells, mediators,and other cells and tissues resident in the airway. Mast cells,inosineophils, epithelial cells, macrophage, and activated T-cells allplay an important role in the inflammatory process associated withasthma. Djukanovic R et al. (1990) Am Rev Respir Dis 142:434-457. It isbelieved that these cells can influence airway function throughsecretion of preformed and newly synthesized mediators which can actdirectly or indirectly on the local tissue. It has also been recognizedthat subpopulations of T-lymphocytes (Th2) play an important role inregulating allergic inflammation in the airway by releasing selectivecytokines and establishing disease chronicity. Robinson D S et al.(1992) N Engl J Med 326:298-304.

Asthma is a complex disorder which arises at different stages indevelopment and can be classified based on the degree of symptoms asacute, subacute or chronic. An acute inflammatory response is associatedwith an early recruitment of cells into the airway. The subacuteinflammatory response involves the recruitment of cells as well as theactivation of resident cells causing a more persistent pattern ofinflammation. Chronic inflammatory response is characterized by apersistent level of cell damage and an ongoing repair process, which mayresult in permanent abnormalities in the airway.

A “subject having asthma” is a subject that has a disorder of therespiratory system characterized by inflammation, narrowing of theairways and increased reactivity of the airways to inhaled agents.Asthma is frequently, although not exclusively, associated with atopicor allergic symptoms. An “initiator” as used herein refers to acomposition or environmental condition which triggers asthma. Initiatorsinclude, but are not limited to, allergens, cold temperatures, exercise,viral infections, SO₂.

The compositions and methods of the invention can be used alone or inconjucnction with other agents and methods useful in the treatment ofasthma. An “asthma/allergy medicament” as used herein is a compositionof matter which reduces the symptoms of, prevents the development of, orinhibits an asthmatic or allergic reaction. Various types of medicamentsfor the treatment of asthma and allergy are described in the GuidelinesFor The Diagnosis and Management of Asthma, Expert Panel Report 2, NIHPublication No. 97/4051, Jul. 19, 1997, the entire contents of which areincorporated herein by reference. The summary of the medicaments asdescribed in the NIH publication is presented below. In most embodimentsthe asthma/allergy medicament is useful to some degree for treating bothasthma and allergy.

Medications for the treatment of asthma are generally separated into twocategories, quick-relief medications and long-term control medications.Asthma patients take the long-term control medications on a daily basisto achieve and maintain control of persistent asthma. Long-term controlmedications include anti-inflammatory agents such as corticosteroids,chromolyn sodium and nedocromil; long-acting bronchodilators, such aslong-acting β₂-agonists and methylxanthines; and leukotriene modifiers.The quick-relief medications include short-acting β₂ agonists,anti-cholinergics, and systemic corticosteroids. There are many sideeffects associated with each of these drugs and none of the drugs aloneor in combination is capable of preventing or completely treatingasthma.

Asthma medicaments include, but are not limited, PDE-4 inhibitors,bronchodilator/beta-2 agonists, K+ channel openers, VLA-4 antagonists,neurokin antagonists, thromboxane A2 (TXA2) synthesis inhibitors,xanthines, arachidonic acid antagonists, 5 lipoxygenase inhibitors, TXA2receptor antagonists, TXA2 antagonists, inhibitor of 5-lipox activationproteins, and protease inhibitors.

Bronchodilator/β₂ agonists are a class of compounds which causebronchodilation or smooth muscle relaxation. Bronchodilator/β₂ agonistsinclude, but are not limited to, salmeterol, salbutamol, albuterol,terbutaline, D2522/formoterol, fenoterol, bitolterol, pirbuerolmethylxanthines and orciprenaline. Long-acting β₂ agonists andbronchodilators are compounds which are used for long-term prevention ofsymptoms in addition to the anti-inflammatory therapies. Long-actingβ₂agonists include, but are not limited to, salmeterol and albuterol.These compounds are usually used in combination with corticosteroids andgenerally are not used without any inflammatory therapy. They have beenassociated with side effects such as tachycardia, skeletal muscletremor, hypokalemia, and prolongation of QTc interval in overdose.

Methylxanthines, including for instance theophylline, have been used forlong-term control and prevention of symptoms. These compounds causebronchodilation resulting from phosphodiesterase inhibition and likelyadenosine antagonism. Dose-related acute toxicities are a particularproblem with these types of compounds. As a result, routine serumconcentration must be monitored in order to account for the toxicity andnarrow therapeutic range arising from individual differences inmetabolic clearance. Side effects include tachycardia, tachyarrhythmias,nausea and vomiting, central nervous system stimulation, headache,seizures, hematemesis, hyperglycemia and hypokalemia. Short-acting β₂agonists include, but are not limited to, albuterol, bitolterol,pirbuterol, and terbutaline. Some of the adverse effects associated withthe administration of short-acting β₂ agonists include tachycardia,skeletal muscle tremor, hypokalemia, increased lactic acid, headache,and hyperglycemia.

Conventional methods for treating or preventing allergy have involvedthe use of anti-histamines or desensitization therapies. Anti-histaminesand other drugs which block the effects of chemical mediators of theallergic reaction help to regulate the severity of the allergic symptomsbut do not prevent the allergic reaction and have no effect onsubsequent allergic responses. Desensitization therapies are performedby giving small doses of an allergen, usually by injection under theskin, in order to induce an IgG-type response against the allergen. Thepresence of IgG antibody helps to neutralize the production of mediatorsresulting from the induction of IgE antibodies, it is believed.Initially, the subject is treated with a very low dose of the allergento avoid inducing a severe reaction and the dose is slowly increased.This type of therapy is dangerous because the subject is actuallyadministered the compounds which cause the allergic response and severeallergic reactions can result.

Allergy medicaments include, but are not limited to, anti-histamines,steroids, and prostaglandin inducers. Anti-histamines are compoundswhich counteract histamine released by mast cells or basophils. Thesecompounds are well known in the art and commonly used for the treatmentof allergy. Anti-histamines include, but are not limited to, astemizole,azelastine, betatastine, buclizine, ceterizine, cetirizine analogues, CS560, desloratadine, ebastine, epinastine, fexofenadine, HSR 609,levocabastine, loratidine, mizolastine, norastemizole, terfenadine, andtranilast.

Prostaglandin inducers are compounds which induce prostaglandinactivity. Prostaglandins function by regulating smooth musclerelaxation. Prostaglandin inducers include, but are not limited to,S-5751.

The asthma/allergy medicaments also include steroids andimmunomodulators. The steroids include, but are not limited to,beclomethasone, fluticasone, triamcinolone, budesonide, corticosteroidsand budesonide.

Corticosteroids include, but are not limited to, beclomethasomedipropionate, budesonide, flunisolide, fluticaosone propionate, andtriamcinolone acetonide. Although dexamethasone is a corticosteroidhaving anti-inflammatory action, it is not regularly used for thetreatment of asthma/allergy in an inhaled form because it is highlyabsorbed and it has long-term suppressive side effects at an effectivedose. Dexamethasone, however, can be used according to the invention forthe treating of asthma/allergy because when administered in combinationwith nucleic acids of the invention it can be administered at a low doseto reduce the side effects. Some of the side effects associated withcorticosteroid include cough, dysphonia, oral thrush (candidiasis), andin higher doses, systemic effects, such as adrenal suppression,osteoporosis, growth suppression, skin thinning and easy bruising.Barnes & Peterson (1993) Am Rev Respir Dis 148:S1-S26; and Kamada A K etal. (1996) Am J Respir Crit Care Med 153:1739-48.

Systemic corticosteroids include, but are not limited to,methylprednisolone, prednisolone and prednisone. Cortosteroids areassociated with reversible abnormalities in glucose metabolism,increased appetite, fluid retention, weight gain, mood alteration,hypertension, peptic ulcer, and aseptic necrosis of bone. Thesecompounds are useful for short-term (3-10 days) prevention of theinflammatory reaction in inadequately controlled persistent asthma. Theyalso function in a long-term prevention of symptoms in severe persistentasthma to suppress and control and actually reverse inflammation. Someside effects associated with longer term use include adrenal axissuppression, growth suppression, dermal thinning, hypertension,diabetes, Cushing's syndrome, cataracts, muscle weakness, and in rareinstances, impaired immune function. It is recommended that these typesof compounds be used at their lowest effective dose (guidelines for thediagnosis and management of asthma; expert panel report to; NIHPublication No. 97-4051; July 1997).

The immunomodulators include, but are not limited to, the groupconsisting of anti-inflammatory agents, leukotriene antagonists, IL-4muteins, soluble IL-4 receptors, immunosuppressants (such as tolerizingpeptide vaccine), anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-IL-9antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, anddownregulators of IgE.

Leukotriene modifiers are often used for long-term control andprevention of symptoms in mild persistent asthma. Leukotriene modifiersfunction as leukotriene receptor antagonists by selectively competingfor LTD-4 and LTE-4 receptors. These compounds include, but are notlimited to, zafirlukast tablets and zileuton tablets. Zileuton tabletsfunction as 5-lipoxygenase inhibitors. These drugs have been associatedwith the elevation of liver enzymes and some cases of reversiblehepatitis and hyperbilirubinemia. Leukotrienes are biochemical mediatorsthat are released from mast cells, inosineophils, and basophils thatcause contraction of airway smooth muscle and increase vascularpermeability, mucous secretions and activate inflammatory cells in theairways of patients with asthma.

Other immunomodulators include neuropeptides that have been shown tohave immunomodulating properties. Functional studies have shown thatsubstance P, for instance, can influence lymphocyte function by specificreceptor-mediated mechanisms. Substance P also has been shown tomodulate distinct immediate hypersensitivity responses by stimulatingthe generation of arachidonic acid-derived mediators from mucosal mastcells. McGillies J et al. (1987) Fed Proc 46:196-9 (1987). Substance Pis a neuropeptide first identified in 1931. Von Euler and Gaddum JPhysiol (London) 72:74-87 (1931). Its amino acid sequence was reportedby Chang et al. in 1971. Chang M M et al. (1971) Nature New Biol232:86-87. The immunoregulatory activity of fragments of substance P hasbeen studied by Siemion I Z et al. (1990) Molec Immunol 27:887-890(1990).

Another class of compounds is the down-regulators of IgE. Thesecompounds include peptides or other molecules with the ability to bindto the IgE receptor and thereby prevent binding of antigen-specific IgE.Another type of downregulator of IgE is a monoclonal antibody directedagainst the IgE receptor-binding region of the human IgE molecule. Thus,one type of downregulator of IgE is an anti-IgE antibody or antibodyfragment. Anti-IgE is being developed by Genentech. One of skill in theart could prepare functionally active antibody fragments of bindingpeptides which have the same function. Other types of IgE downregulatorsare polypeptides capable of blocking the binding of the IgE antibody tothe Fc receptors on the cell surfaces and displacing IgE from bindingsites upon which IgE is already bound.

One problem associated with downregulators of IgE is that many moleculesdo not have a binding strength to the receptor corresponding to the verystrong interaction between the native IgE molecule and its receptor. Themolecules having this strength tend to bind irreversibly to thereceptor. However, such substances are relatively toxic since they canbind covalently and block other structurally similar molecules in thebody. Of interest in this context is that the α chain of the IgEreceptor belongs to a larger gene family where, e.g., several of thedifferent IgG Fc receptors are contained. These receptors are absolutelyessential for the defense of the body against, e.g., bacterialinfections. Molecules activated for covalent binding are, furthermore,often relatively unstable and therefore they probably have to beadministered several times a day and then in relatively highconcentrations in order to make it possible to block completely thecontinuously renewing pool of IgE receptors on mast cells and basophilicleukocytes.

Chromolyn sodium and nedocromil are used as long-term controlmedications for preventing primarily asthma symptoms arising fromexercise or allergic symptoms arising from allergens. These compoundsare believed to block early and late reactions to allergens byinterfering with chloride channel function. They also stabilize mastcell membranes and inhibit activation and release of mediators frominosineophils and epithelial cells. A four to six week period ofadministration is generally required to achieve a maximum benefit.

Anticholinergics are generally used for the relief of acutebronchospasm. These compounds are believed to function by competitiveinhibition of muscarinic cholinergic receptors. Anticholinergicsinclude, but are not limited to, ipratropium bromide. These compoundsreverse only cholinerigically-mediated bronchospasm and do not modifyany reaction to antigen. Side effects include drying of the mouth andrespiratory secretions, increased wheezing in some individuals, andblurred vision if sprayed in the eyes.

In addition to standard asthma/allergy medicaments, other methods fortreating asthma/allergy have been used either alone or in combinationwith established medicaments. One preferred, but frequently impossible,method of relieving allergies is allergen or initiator avoidance.Another method currently used for treating allergic disease involves theinjection of increasing doses of allergen to induce tolerance to theallergen and to prevent further allergic reactions.

Allergen injection therapy (allergen immunotherapy) is known to reducethe severity of allergic rhinitis. This treatment has been theorized toinvolve the production of a different form of antibody, a protectiveantibody which is termed a “blocking antibody”. Cooke R A et al. (1935)Serologic Evidence of Immunity with Coexisting Sensitization in a Typeof Human Allergy, Exp Med 62:733. Other attempts to treat allergyinvolve modifying the allergen chemically so that its ability to causean immune response in the patient is unchanged, while its ability tocause an allergic reaction is substantially altered. These methods,however, can take several years to be effective and are associated withthe risk of side effects such as anaphylactic shock.

The compositions and methods of the invention can be used to modulate animmune response. The ability to modulate an immune response allows forthe prevention and/or treatment of particular disorders that can beaffected via immune system modulation.

Treatment after a disorder has started aims to reduce, ameliorate, oraltogether eliminate the disorder, and/or its associated symptoms, orprevent it from becoming worse. Treatment of subjects before a disorderhas started (i.e., prophylactic treatment) aims to reduce the risk ofdeveloping the disorder. As used herein, the term “prevent” refers tothe prophylactic treatment of patients who are at risk of developing adisorder (resulting in a decrease in the probability that the subjectwill develop the disorder), and to the inhibition of further developmentof an already established disorder.

Different doses may be necessary for treatment of a subject, dependingon activity of the compound, manner of administration, purpose of theimmunization (i.e., prophylactic or therapeutic), nature and severity ofthe disorder, age and body weight of the subject. The administration ofa given dose can be carried out both by single administration in theform of an individual dose unit or else several smaller dose units.Multiple administration of doses at specific intervals of weeks ormonths apart is usual for boosting antigen-specific immune responses.

Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat theparticular subject. The effective amount for any particular applicationcan vary depending on such factors as the disease or condition beingtreated, the particular therapeutic agent being administered (e.g., inthe case of an immunostimulatory nucleic acid, the type of nucleic acid,i.e., a CpG nucleic acid, the number of unmethylated CpG motifs or theirlocation in the nucleic acid, the degree of modification of the backboneto the oligonucleotide, etc.), the size of the subject, or the severityof the disease or condition. One of ordinary skill in the art canempirically determine the effective amount of a particular nucleic acidand/or other therapeutic agent without necessitating undueexperimentation.

Subject doses of the compounds described herein typically range fromabout 0.1 μg to 10,000 mg, more typically from about 1 μg/day to 8000mg, and most typically from about 10 μg to 100 μg. Stated in terms ofsubject body weight, typical dosages range from about 0.1 μg to 20mg/kg/day, more typically from about 1 to 10 mg/kg/day, and mosttypically from about 1 to 5 mg/kg/day.

The pharmaceutical compositions containing nucleic acids and/or othercompounds can be administered by any suitable route for administeringmedications. A variety of administration routes are available. Theparticular mode selected will depend, of course, upon the particularagent or agents selected, the particular condition being treated, andthe dosage required for therapeutic efficacy. The methods of thisinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of an immune response without causingclinically unacceptable adverse effects. Preferred modes ofadministration are discussed herein. For use in therapy, an effectiveamount of the nucleic acid and/or other therapeutic agent can beadministered to a subject by any mode that delivers the agent to thedesired surface, e.g., mucosal, systemic.

Administering the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Routes ofadministration include but are not limited to oral, parenteral,intravenous, intramuscular, intranasal, sublingual, intratracheal,inhalation, subcutaneous, ocular, vaginal, and rectal. For the treatmentor prevention of asthma or allergy, such compounds are preferablyinhaled, ingested or administered by systemic routes. Systemic routesinclude oral and parenteral. Inhaled medications are preferred in someembodiments because of the direct delivery to the lung, the site ofinflammation, primarily in asthmatic patients. Several types of devicesare regularly used for administration by inhalation. These types ofdevices include metered dose inhalers (MDI), breath-actuated MDI, drypowder inhaler (DPI), spacer/holding chambers in combination with MDI,and nebulizers.

The therapeutic agents of the invention may be delivered to a particulartissue, cell type, or to the immune system, or both, with the aid of avector. In its broadest sense, a “vector” is any vehicle capable offacilitating the transfer of the compositions to the target cells. Thevector generally transports the immunostimulatory nucleic acid,antibody, antigen, and/or disorder-specific medicament to the targetcells with reduced degradation relative to the extent of degradationthat would result in the absence of the vector.

In general, the vectors useful in the invention are divided into twoclasses: biological vectors and chemical/physical vectors. Biologicalvectors and chemical/physical vectors are useful in the delivery and/oruptake of therapeutic agents of the invention.

Most biological vectors are used for delivery of nucleic acids and thiswould be most appropriate in the delivery of therapeutic agents that areor that include immunostimulatory nucleic acids.

In addition to the biological vectors discussed herein,chemical/physical vectors may be used to deliver therapeutic agentsincluding immunostimulatory nucleic acids, antibodies, antigens, anddisorder-specific medicaments. As used herein, a “chemical/physicalvector” refers to a natural or synthetic molecule, other than thosederived from bacteriological or viral sources, capable of delivering thenucleic acid and/or other medicament.

A preferred chemical/physical vector of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvesicles (LUVs), which range in size from 0.2-4.0 μm can encapsulatelarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form. Fraley et al. (1981) Trends Biochem Sci 6:77.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Ligands which may be useful for targeting aliposome to an immune cell include, but are not limited to: intact orfragments of molecules which interact with immune cell specificreceptors and molecules, such as antibodies, which interact with thecell surface markers of immune cells. Such ligands may easily beidentified by binding assays well known to those of skill in the art. Instill other embodiments, the liposome may be targeted to the cancer bycoupling it to a one of the immunotherapeutic antibodies discussedearlier. Additionally, the vector may be coupled to a nuclear targetingpeptide, which will direct the vector to the nucleus of the host cell.

Lipid formulations for transfection are commercially available fromQIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a specialDNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimerictechnology).

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3 dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis G(1985) Trends Biotechnol 3:235-241.

In one embodiment, the vehicle is a biocompatible microparticle orimplant that is suitable for implantation or administration to themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO95/24929, entitled“Polymeric Gene Delivery System”. PCT/US/0307 describes a biocompatible,preferably biodegradable polymeric matrix for containing an exogenousgene under the control of an appropriate promoter. The polymeric matrixcan be used to achieve sustained release of the therapeutic agent in thesubject.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein the nucleic acid and/or the other therapeuticagent is dispersed throughout a solid polymeric matrix) or amicrocapsule (wherein the nucleic acid and/or the other therapeuticagent is stored in the core of a polymeric shell). Other forms of thepolymeric matrix for containing the therapeutic agent include films,coatings, gels, implants, and stents. The size and composition of thepolymeric matrix device is selected to result in favorable releasekinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method ofdelivery which is to be used, typically injection into a tissue oradministration of a suspension by aerosol into the nasal and/orpulmonary areas. Preferably when an aerosol route is used the polymericmatrix and the nucleic acid and/or the other therapeutic agent areencompassed in a surfactant vehicle.

The polymeric matrix composition can be selected to have both favorabledegradation rates and also to be formed of a material which isbioadhesive, to further increase the effectiveness of transfer when thematrix is administered to a nasal and/or pulmonary surface that hassustained an injury. The matrix composition also can be selected not todegrade, but rather, to release by diffusion over an extended period oftime. In some preferred embodiments, the nucleic acid are administeredto the subject via an implant while the other therapeutic agent isadministered acutely. Biocompatible microspheres that are suitable fordelivery, such as oral or mucosal delivery, are disclosed in Chickeringet al. (1996) Biotech Bioeng 52:96-101 and Mathiowitz E et al. (1997)Nature 386:410-414 and PCT Pat. Application WO97/03702.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the nucleic acid and/or the other therapeutic agent to thesubject. Biodegradable matrices are preferred. Such polymers may benatural or synthetic polymers. The polymer is selected based on theperiod of time over which release is desired, generally in the order ofa few hours to a year or longer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable, particularly for the nucleic acid agents. The polymeroptionally is in the form of a hydrogel that can absorb up to about 90%of its weight in water and further, optionally is cross-linked withmulti-valent ions or other polymers.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein. These include polyhyaluronic acids, casein,gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan,poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate).

If the therapeutic agent is a nucleic acid, the use of compaction agentsmay also be desirable. Compaction agents also can be used alone, or incombination with, a biological or chemical/physical vector. A“compaction agent”, as used herein, refers to an agent, such as ahistone, that neutralizes the negative charges on the nucleic acid andthereby permits compaction of the nucleic acid into a fine granule.Compaction of the nucleic acid facilitates the uptake of the nucleicacid by the target cell. The compaction agents can be used alone, i.e.,to deliver a nucleic acid in a form that is more efficiently taken up bythe cell or, more preferably, in combination with one or more of theabove-described vectors.

Other exemplary compositions that can be used to facilitate uptake of anucleic acid include calcium phosphate and other chemical mediators ofintracellular transport, microinjection compositions, electroporationand homologous recombination compositions (e.g., for integrating anucleic acid into a preselected location within the target cellchromosome).

The compounds may be administered alone (e.g., in saline or buffer) orusing any delivery vectors known in the art. For instance the followingdelivery vehicles have been described: cochleates (Gould-Fogerite etal., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 1997);ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Moreinet al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989,1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella,Escherichia coli, Bacillus calmatte-guerin, Shigella, Lactobacillus)(Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stoveret al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia,adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al.,1996, Nugent et al., 1998, Flexner et al., 1988, Morrow et al., 1999);microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al.,1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989);nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasakiet al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g.carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill etal., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott etal., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi etal., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998,Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al.,1995, Cryz et al., 1998); and, virus-like particles (Jiang et al., 1999,Leibl et al., 1998).

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

The term pharmaceutically-acceptable carrier means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration to a human or other vertebrateanimal. The term carrier denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being commingled with the compounds ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficiency.

For oral administration, the compounds (i.e., nucleic acids, antigens,antibodies, and other therapeutic agents) can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer R (1990) Science249:1527-1533, which is incorporated herein by reference.

The nucleic acids and optionally other therapeutics and/or antigens maybe administered per se (neat) or in the form of a pharmaceuticallyacceptable salt. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-, di-, and tri-glycerides; hydrogelrelease systems; silastic systems; peptide-based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the invention iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

The invention also provides efficient methods of identifyingimmunostimulatory compounds and optimizing the compounds and agents soidentified. Generally, the screening methods involve assaying forcompounds which inhibit or enhance signaling through a particular TLR.The methods employ a TLR, a suitable reference ligand for the TLR, and acandidate immunostimulatory compound. The selected TLR is contacted witha suitable reference compound (TLR ligand) and a TLR-mediated referencesignal is measured. The selected TLR is also contacted with a candidateimmunostimulatory compound and a TLR-mediated test signal is measured.The test signal and the reference signal are then compared. A favorablecandidate immunostimulatory compound may subsequently be used as areference compound in the assay. Such methods are adaptable toautomated, high throughput screening of candidate compounds. Examples ofsuch high throughput screening methods are described in U.S. Pat. Nos.6,103,479; 6,051,380; 6,051,373; 5,998,152; 5,876,946; 5,708,158;5,443,791; 5,429,921; and 5,143,854.

As used herein “TLR signaling” refers to an ability of a TLR polypeptideto activate the Toll/IL-1R (TIR) signaling pathway, also referred toherein as the TLR signal transduction pathway. Changes in TLR activitycan be measured by assays designed to measure expression of genes undercontrol of κB-sensitive promoters and enhancers. Such genes can benaturally occurring genes or they can be genes artificially introducedinto a cell. Naturally occurring reporter genes include the genesencoding IL-1β, IL-6, IL-8, the p40 subunit of interleukin 12 (IL-12p40), and the costimulatory molecules CD80 and CD86. Other genes can beplaced under the control of such regulatory elements and thus serve toreport the level of TLR signaling.

The assay mixture comprises a candidate immunostimulatory compound.Typically, a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a different response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateimmunostimulatory compounds may encompass numerous chemical classes,although typically they are organic compounds. In some embodiments, thecandidate immunostimulatory compounds are small RNAs or small organiccompounds, i.e., organic compounds having a molecular weight of morethan 50 yet less than about 2500 Daltons. Polymeric candidateimmunostimulatory compounds can have higher molecular weights, e.g.,oligonucleotides in the range of about 2500 to about 12,500. Candidateimmunostimulatory compounds also may be biomolecules such as nucleicacids, peptides, saccharides, fatty acids, sterols, isoprenoids,purines, pyrimidines, derivatives or structural analogs of the above, orcombinations thereof and the like. Where the candidate immunostimulatorycompound is a nucleic acid, the candidate immunostimulatory compoundtypically is a DNA or RNA molecule, although modified nucleic acidshaving non-natural bonds or subunits are also contemplated.

Candidate immunostimulatory compounds may be obtained from a widevariety of sources, including libraries of natural, synthetic, orsemisynthetic compounds, or any combination thereof. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily modified through conventional chemical, physical, andbiochemical means. Further, known pharmacological agents may besubjected to directed or random chemical modifications such asacylation, alkylation, esterification, amidification, etc., to producestructural analogs of the candidate immunostimulatory compounds.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc., which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The order of addition of components, incubation temperature, time ofincubation, and other parameters of the assay may be readily determined.Such experimentation merely involves optimization of the assayparameters, not the fundamental composition of the assay. Incubationtemperatures typically are between 4° C. and 40° C., more typicallyabout 37° C. Incubation times preferably are minimized to facilitaterapid, high throughput screening, and typically are between 1 minute and10 hours.

After incubation, the level of TLR signaling is detected by anyconvenient method available to the user. For cell-free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. For example, separation can be accomplished in solution, or,conveniently, at least one of the components is immobilized on a solidsubstrate, from which the unbound components may be easily separated.The solid substrate can be made of a wide variety of materials and in awide variety of shapes, e.g., microtiter plate, microbead, dipstick,resin particle, etc. The substrate preferably is chosen to maximizesignal-to-noise ratios, primarily to minimize background binding, aswell as for ease of separation and cost.

Separation may be effected, for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatographic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, non-specific protein, etc. Where the solid substrateis a magnetic bead, the beads may be washed one or more times with awashing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as measurement of an induced polypeptide within, on the surface of,or secreted by the cell. Examples of detection methods useful incell-based assays include fluorescence-activated cell sorting (FACS)analysis, bioluminescence, fluorescence, enzyme-linked immunosorbentassay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR),and the like. Examples of detection methods useful in cell-free assaysinclude bioluminescence, fluorescence, ELISA, RT-PCR, and the like.

EXAMPLES Example 1 Responsiveness of Human PBMC to G,U-ContainingOligoribonucleotides

Human peripheral blood mononuclear cells (PBMCs) were isolated fromhealthy donors, plated at 3×10⁵ cells/well, stimulated in vitro withvarious test and control immunostimulatory agents for 16 hours, and thenanalyzed by enzyme-linked immunosorbent assay (ELISA) using matchedantibody pairs from BD-Pharmingen for secreted cytokines IL-12 p40 andTNF-α, performed according to the manufacturer's protocol. Also includedwere certain negative controls, including medium alone and DOTAP (10μg/200 μl culture well; “Liposomes”) alone. The controlimmunostimulatory agents included the imidazoquinolone R-848 (2 μg/ml),lipopolysaccharide (LPS; 1 μg/ml), Pam3Cys (5 μg/ml), poly IC (50μg/ml), and CpG DNA (50 μg/ml). These are reported ligands for TLR7,TLR4, TLR2, TLR3, and TLR9, respectively. Test immunostimulatory agentsincluded the following RNA molecules, each at 50 μg/ml, with and withoutDOTAP (10 μg total “with Liposomes” and “without Liposomes”,respectively): GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combinationwith GUAGGCAC; GUAGGA; GAAGGCAC; CUAGGCAC; CUCGGCAC; and CCCCCCCC. TheseRNA oligonucleotides each contained a phosphorothioate linkage betweenthe penultimate and 3′ terminal nucleoside.

FIG. 1 depicts the responsiveness of human PBMC to the test and controlagents listed above, as measured by secreted amounts of IL-12 p40(pg/ml). As can be seen in FIG. 1, PBMCs were responsive to R-848, LPS,Pam3Cys, and poly IC, while they were unresponsive to DOTAP alone.Significantly, human PBMC secreted large amounts of IL-12 p40 (10-20ng/ml) in response to G,U-containing RNA oligonucleotides GUGUUUACalone; GUAGGCAC alone; GUGUUUAC in combination with GUAGGCAC; CUAGGCAC;and CUCGGCAC, each in combination with DOTAP. Also significantly, humanPBMC did not secrete significant amounts of IL-12 p40 in response toG,U-free RNA oligonucleotides GAAGGCAC and CCCCCCCC. Theimmunostimulatory effect of the G,U-containing RNA molecules appeared tobe greatly enhanced by the inclusion of DOTAP. In this experiment, theG,U-containing 6-mer RNA GUAGGA appeared to exert little, if anyimmunostimulatory effect either with or without DOTAP.

FIG. 2 depicts the responsiveness of human PBMC to the test and controlagents listed above, as measured by secreted amounts of TNF-α. A similarpattern of results was observed as in FIG. 1, i.e., human PBMC secretedlarge amounts of TNF-α (40-100 ng/ml) in response to G,U-containing RNAoligonucleotides GUGUUUAC alone; GUAGGCAC alone; GUGUUUAC in combinationwith GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP.Also similar to the results in FIG. 1, human PBMC did not secretesignificant amounts of TNF-α in response to G,U-free RNAoligonucleotides GAAGGCAC and CCCCCCCC, or in response to theG,U-containing 6-mer RNA GUAGGA. The immunostimulatory effect of theG,U-containing RNA molecules appeared to be greatly enhanced by theinclusion of DOTAP.

It will be appreciated in this example that the following partialself-complementarity basepairing is possible, where G-U wobble basepairsare shown joined with a dot and G-C and A-U basepairs are shown joinedby a line:

Example 2 Dose-Response Behavior of Human PBMC to G,U-ContainingOligoribonucleotides

The experiments described in the preceding example were repeated withvaried concentrations of RNA oligonucleotides in order to assess thedose-response behavior of human PBMCs to G,U-containing RNAoligonucleotides of the invention. A total of 10, 3 or 1 μg RNA wasadded to 10 μg DOTAP and then added to the 200 μl culture wells. After16 hours IL-12 p40 and TNF-α ELISAs were performed as described inExample 1.

FIG. 3 depicts the dose-response of human PBMC to the various RNAs asmeasured by secreted amounts of IL-12 p40 (ng/ml). As can be seen fromFIG. 3, human PBMC secreted increasing amounts of IL-12 p40 in responseto increasing amounts of G,U-containing RNA oligomers GUGUUUAC;GUAGGCAC; CUAGGCAC; and CUCGGCAC, each in combination with DOTAP.Conversely, FIG. 3 also shows that human PBMC appeared not to secreteIL-12 p40 in response to any of the tested amounts of G,U-free RNAoligomers GAAGGCAC or CCCCCCCC.

Corresponding dose-response of human PBMC to the various RNAs wasmeasured by secreted amounts of TNF-α. A similar pattern of results wasobserved as in FIG. 3, i.e., human PBMC secreted increasing amounts ofTNF-α in response to increasing amounts G,U-containing RNAoligonucleotides GUGUUUAC; GUAGGCAC; CUAGGCAC; and CUCGGCAC, each incombination with DOTAP. Also similar to the results in FIG. 3, humanPBMC did not appear to secrete significant amounts of TNF-α in responseto any of the tested amounts of G,U-free RNA oligonucleotides GAAGGCACand CCCCCCCC.

Example 3 Base Sequence Sensitivity of RNA Oligomers

Point mutations were made to the RNA oligonucleotide GUAGGCAC bysubstituting A or C at selected positions. The variousoligoribonucleotides included the following: GUAGGCAC; GUAGGA; GAAGGCAC;AUAAACAC; AUAGACAC; AUAAGCAC; GUAAACAC; CUAGGCAC; CUCGGCAC; andGUGUUUAC. The oligonucleotides were titrated onto human PBMC isolatedfrom healthy donors and plated at 3×10⁵ cells/well. A total of 10 μg RNAwas added to 10 μg DOTAP and then added to the 200 μl culture wells.Human TNF-α was measured by ELISA using matched antibody pairs fromBD-Pharmingen according to the manufacturer's protocol. Results areshown in FIG. 4.

Example 4 Effect of DOTAP on Human PBMC Response to Various Stimuli

In order to characterize further the role of DOTAP in theimmunostimulatory effects of the G,U-containing RNA oligomers observedin the previous examples, human PBMCs were isolated from healthy donors,plated at 3×10⁵ cells/well, and stimulated in the presence of known TLRligands, either with or without DOTAP (“with Liposomes” or “withoutLiposomes”, respectively). The known TLR ligands examined were total RNAprepared from hyphae (hyphae), total RNA prepared from yeast (yeast),total RNA prepared from promyelocytic cell line HL-60 (HL60), in vitrotranscribed ribosomal RNA for E. coli Sp6, in vitro transcribedribosomal RNA for E. coli T7, LPS, poly IC, Pam3Cys, and R-848. Mediumalone and DOTAP alone were used as negative controls. The panel of RNAsfrom the previous examples, again at 10 μg/ml and without DOTAP, wasalso included.

Total RNA was isolated from the human promyelocytic cell line HL-60using Trizol (Sigma). Prior to isolation, cells were treated for 4 hourswith 500 μM hydrogen peroxide (H₂O₂), which induces apoptosis in thiscell line (HL60 500). Untreated cells served as control (HL60 0).

Candida albicans RNA was isolated from yeast or hyphae (induced by 4 hincubation with 10% fetal calf serum). Cells from a 100 ml culture werepelleted, washed and resuspended in 10 ml of Tris/EDTA buffer (10 mM, 1mM). RNA was isolated by extraction with hot acidic phenol according tomethods described in Ausubel F M et al., eds., Current Protocols inMolecular Biology, John Wiley & Sons, New York.

The genomic fragment of E. coli 16S RNA was amplified with the primers5′-ATTGAAGAGTTTGATCATGGCTCAGATTGAACG-3′ (SEQ ID NO:5) and5′-TAAGGAGGTGATCCAACCGCAGGTTCC-3′ (SEQ ID NO:6) from genomic E. coli DNAand cloned into the pGEM T easy vector. In vitro transcription wasperformed using T7 or Sp6 RNA polymerase. Transcribed RNA was furtherpurified by chloroform/phenol extraction, precipitated, and used at 10μg.

Following 16 hour incubation, ELISAs were performed as before to assesssecretion of IL-12 p40 and TNF-α. Representative results are shown inFIG. 5.

FIG. 5 depicts the effect of DOTAP on the amount of IL-12 p40 secretedby human PBMC following incubation with and without DOTAP. As can beseen from the figure, the following stimuli appeared to exert greaterimmunostimultory effect in the presence of DOTAP than in its absence:hyphae, yeast, E. coli Sp6, and E. coli T7. The following stimuliappeared to exert reduced immunostimultory effect in the presence ofDOTAP than in its absence: LPS, poly IC. The following stimuli appearedto exert about the same immunostimultory effect in the presence orabsence of DOTAP: HL60, Pam3Cys and R-848.

Example 5 Immunostimulatory Effect of G,U-Containing RNA Oligomers isSpecies- and MyD88-Dependent

The following murine cells were isolated and incubated with various RNAsand other known TLR ligands in order to assess species-, cell type-, andsignaling pathway-specificity: wild type macrophages in the presence ofIFN-γ; MyD88-deficient macrophages in the presence of IFN-γ; J774 (mousemacrophage cell line); and RAW 264.7 (mouse macrophage cell line, e.g.,ATCC TIB-71). Murine bone macrophages were generated from wild type orMyD88-deficient C57BL/6 mice by culturing bone marrow cells with 50ng/ml M-CSF for 5 days. Cells were seeded at 25,000 cells/well andtreated with 20 ng/ml IFN-γ for 16 hours. The murine macrophage celllines RAW and J774 were seeded at 10,000 cells/well.

The following test and control agents were examined: R-848 (2 μg/ml),ODN 1668 (CpG DNA; 5′-TCCATGACGTTCCTGATGCT-3′; SEQ ID NO:7); LPS (1μg/ml); poly IC (50 μg/ml); Pam3Cys (5 μg/ml); Ionomycin/TPA; thefollowing RNA molecules, each with (“+ Lipo”) and without DOTAP (10μg/200 μl culture well): GUGUUUAC alone (RNA1); GUAGGCAC alone (RNA2);GUGUUUAC in combination with GUAGGCAC (RNA1/2);UCCGCAAUGGACGAAAGUCUGACGGA (RNA6; SEQ ID NO:8);GAGAUGGGUGCGAGAGCGUCAGUAUU (RNA9; SEQ ID NO:9); and the following DNAmolecules, corresponding to RNA1, RNA2, and RNA1/2: GTGTTTAC alone(DNA1); GTAGGCAC alone (DNA2); and GTGTTTAC in combination with GTAGGCAC(DNA1/2). These RNA and DNA oligonucleotides each contained aphosphorothioate linkage between the penultimate and 3′ terminalnucleoside. RNA6 and RNA9 each contained in addition a phosphorothioatelinkage between the penultimate and 5′ terminal nucleoside. RNA6corresponds to a ribosomal RNA stem loop derived from Listeriamonocytogenes. RNA9 corresponds to a stem loop derived from humanimmunodeficiency virus (HIV, an RNA retrovirus). The cells were culturedfor 12 hours and supernatants were harvested. Murine IL-12 p40, IL-6,and TNF-α were measured by ELISA using matched antibody pairs fromBD-Pharmingen according to the manufacturer's protocol. Representativeresults are shown in FIG. 6.

Panel A of FIG. 6 shows that wild type murine macrophages in thepresence of IFN-γ secrete significant amounts of IL-12 p40 in responseto R-848; ODN 1668 (CpG DNA); LPS; poly IC; Pam3Cys; and G,U-containingRNA oligomers GUGUUUAC in combination with GUAGGCAC (with DOTAP). Incontrast, Panel B of FIG. 6 shows that MyD88-deficient murinemacrophages in the presence of IFN-γ secrete little or no IL-12 p40 inresponse to any of the test and control agents examined, thusdemonstrating a dependence on MyD88 for immunostimulatory response tothese compounds. Such a result is consistent with participation by a TLRin the immunostimulatory response to any of these compounds, includingin particular the G,U-containing RNA oligonucleotides of the invention.Panels C and D of FIG. 6 show generally similar, if somewhat attenuated,response patterns of J774 and RAW 264.7 mouse macrophage cell lines asfor wild type murine macrophages in the presence of IFN-γ, as shown inPanel A. Essentially similar results were found in parallel ELISAsmeasuring IL-6 and TNF-α.

In additional studies involving MyD88 wild-type cells, it was observedthat addition of bafilomycin largely or completely abrogated theimmunostimulatory effect of the RNA oligomers. Together with theMyD88-dependence, this observation is consistent with involvement of atleast one of TLR3, TLR7, TLR8, and TLR9.

Example 6 Use of Cholesteryl Ester in Place of Cationic Lipid

In order to investigate the possibility of using cholesterylester-modified RNA oligomer in place of RNA oligomer plus cationiclipid, RNA oligomer GUGUGUGU was prepared with (R 1058) and without (R1006) a 3′ cholesteryl ester modification. These two RNA oligomers withand without DOTAP, were added over a range of concentrations toovernight cultures of human PBMC. Culture supernatants were harvested,and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA usingmatched antibody pairs from BD-Pharmingen according to themanufacturer's protocol. Representative results for experimentsincluding DOTAP are shown in Table 1.

TABLE 1 Cholesteryl Ester Modification in Place of DOTAP TNF-α TNF-αIFN-α IFN-α +DOTAP −DOTAP +DOTAP −DOTAP EC50 max EC50, max EC50 max EC50max ID μM pg/ml μM pg/ml μM pg/ml μM pg/ml R 1006 2.8 40000 7.8 2200 4.55000 — — R 1058 0.2 75000 1.0 3000 0.5 3800 0.5 1500The results indicate that R 1058, with the cholesteryl estermodification, is more potent than R 1006, having the same base sequencebut without cholesterol, both with and without DOTAP.

Example 7 Effect of Oligomer Length

RNA oligomers GUGUGUGU, GUGUGUG, GUGUGU, GUGUG, GUGU, GUG, and GU, withand without DOTAP, were added over a range of concentrations toovernight cultures of human PBMC. Culture supernatants were harvested,and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA usingmatched antibody pairs from BD-Pharmingen according to themanufacturer's protocol. Representative results for experimentsincluding DOTAP are shown in Table 2.

TABLE 2 Effect of RNA Oligomer Length TNF-α IL-12 p40 IFN-α ID SEQEC50, μM max pg/ml EC50, μM max pg/ml EC50, μM max pg/ml R 1006 GUGUGUGU2.8 40000 1.6 7000 4.5 5000 R 1048 GUGUGUG 2.2 30000 2.6 10000 4.6 2700R 1049 GUGUGU 6.7 30000 2.1 8000 4.8 3400 R 1050 GUGUG 7.6 40000 3.914000 6.9 400 R 1051 GUGU — — >20 14000 — — R 1052 GUG — — >20 6000 5.5800 R 1053 GU — — >20 5000 — —

Example 8 Effect of Stabilization of Internucleoside Linkages

GUGUGUGU RNA oligomers were synthesized with specific phosphorothioateand phosphodiester linkages as shown in Table 2, where “*” representsphosphorothioate and “_” represents phosphodiester. RNA oligomers, withand without DOTAP, were added over a range of concentrations toovernight cultures of human PBMC. Culture supernatants were harvested,and human TNF-α, IL-12 p40, and IFN-α were measured by ELISA usingmatched antibody pairs from BD-Pharmingen according to themanufacturer's protocol. Representative results for experimentsincluding DOTAP are shown in Table 3.

TABLE 3 UZ,4/36 Effect of Stabilization of Internucleoside LinkagesTNF-α IFN-α ID SEQ EC50, μM max, pg/ml EC50, μM max, pg/ml R 1006G*U*G*U*G*U*G*U 2.8 40000 4.5 5000 R 1054 G*U_G*U*G*U*G*U 5.6 40000 6.73700 R 1055 G*U_G*U_G*U*G*U >20 20000 — — R 1056 G*U_G*U_G*U_G*U >2012000 — — R 1057 G_U_G_U_G_U_G_U — — 0.1 6000

In like manner, an all-phosphodiester 40-mer capable of forming astem-loop structure and having a base sequence as provided by5′-CACACACUGCUUAAGCGCUUGCCUGCUUAAGUAGUGUGUG-3′ (R 1041; SEQ ID NO:10)was synthesized and tested in overnight culture with human PBMC. ThisRNA oligomer was found to be very potent in its ability to induce IFN-α,with an EC50 of <0.1 μM and a maximum of 5000 pg/ml.

Example 9 DNA:RNA Conjugates

A series of DNA:RNA conjugates, each containing the RNA sequenceGUGUGUGU and a poly-dT or a poly-dG sequence, was prepared. Theoligomers were as follows, where again “*” represents phosphorothioateand “_” represents phosphodiester:

(R 1060; SEQ ID NO:11) G*U*G*U*G*U*G*U_dG_dG*dG*dG*dG*dG (R 1061; SEQ ID NO:12) dG*dG*dG*dG_dG_G*U*G*U*G*U*G*U (R 1062; SEQ ID NO:13) G*U*G*U*G*U*G*U*dT*dT*dT*dT*dT*dT (R 1063; SEQ ID NO:14) dT*dT*dT*dT*dT*G*U*G*U*G*U*G*U Human PBMC were cultured overnight in the presence of added DNA:RNAconjugate, with and without DOTAP. Culture supernatants were harvestedand human TNF-α, IL-6, IL-12 p40, IP-10, and IFN-α were measured byELISA using matched antibody pairs from BD-Pharmingen according to themanufacturer's protocol. Representative results for experimentsincluding DOTAP are shown in Table 4.

TABLE 4 Immunostimulatory DNA:RNA Conjugates TNF-α IL-6 IP-10 EC50,EC50, EC50, ID μM max pg/ml μM max pg/ml μM max pg/ml R 1060 4.9 20000 —— — — R 1061 4.3 20000 >20 10000 1.1 180 R 1062 0.3 80000 0.4 28000 0.1400 R 1063 0.3 60000 0.8 28000 0.1 250

Example 10 Transfer RNA

Human PBMC were cultured overnight in the presence of variousconcentrations (1, 3, and 10 μg/ml) of tRNA obtained from wheat germ,bovine, yeast, and E. coli sources, added to the culture medium with andwithout DOTAP. Culture supernatants were harvested and human TNF-α andIL-12 p40 were measured by ELISA using matched antibody pairs fromBD-Pharmingen according to the manufacturer's protocol. Yeast and E.coli tRNAs, and to a lesser extent bovine tRNA, induced TNF-α and IL-12p40 when DOTAP was also present. In addition, E. coli tRNA at 3 and 10μg/ml induced minor amounts of both cytokines even without DOTAP.

Example 11 HIV RNA

Hunan PBMC were incubated overnight with either of two key G,U-richsequences, namely 5′-GUAGUGUGUG-3′ (SEQ ID NO:2) and 5′-GUCUGUUGUGUG-3′(SEQ ID NO:3), corresponding to nt 99-108 and 112-123 of HIV-1 strainBH10, respectively, each with and without DOTAP. Culture supernatantswere harvested, and human IL-12 p40 and TNF-α were measured by ELISAusing matched antibody pairs from BD-Pharmingen according to themanufacturer's protocol. Representative results are shown in FIG. 7. Thefigure shows that both of these RNA molecules, at micromolarconcentrations in the presence of DOTAP, induced 50-100 ng/ml of TNF and50-200 ng/ml of IL-12 p40.

Example 12 Responsiveness of Human PBMC to Stringent Response Factor

When bacteria are starved they enter into a programmed response termedthe stringent response. This involves the production of nucleic acidalarmones and ribosomal loss. Bacteria growing at high rates contain70,000-80,000 ribosomes accounting for as much as 50% of their dryweight. As growth slows, unneeded ribosomes are hydrolyzed. It washypothesized that rapidly growing cells in their early stationary phasecontain large amounts of oligoribonucleotides that are released into themedia when the cells enter a neutral pH environment.

FIG. 10 depicts the responsiveness of human PBMC to stringent responsefactor (SRF). SRF is produced by rapidly growing bacteria (in this caseListeria monocytogenes) in rich media until their late log phase. Thebacteria were pelleted and resuspended in an equal volume of PBS for 24h. The mixture is centrifuged to remove the bacteria. The supernatant issterilized by passing it through a 0.2 μm filter. The sterilizedsolution was passed through a molecular filter with a cutoff of 10 kDa.This fraction was separated on a C18 column and the eluant was tested.At a concentration of 5 μg/ml SRF induced TNF from human PBMC. If SRFwas treated with any of three RNAses the activity was destroyed. Theactivity was not due to substances other than RNA because theRNase-treated SRF had near background stimulatory ability. This impliedactivity was due to RNA.

Example 13 Responsiveness of Human PBMC to Ribonucleoside VanadylComplexes

During studies of SRF it was surprisingly determined that the RNAseinhibitor, ribonucleoside vanadyl complexes (RVCs), could stimulatehuman PBMC to produce TNF (FIG. 11) and IL-6.

FIG. 11 depicts the responsiveness of human PBMC to the ribonucleosidevanadyl complexes (RVCs). It was unexpectedly discovered during testingof RNAse inhibitors that RVCs were stimulatory for human PBMC. 2 mM RVCinduced the release of substantial TNF. Also tested was the anti-viralimidazoquinoline, resiquimod (R-848) denoted as X and used at 0.1 μg/ml.

Example 14 Responsiveness of Human TLR7 and Human TLR8 toRibonucleosides

The observations of Example 13 could be extended to 293 cellsgenetically reconstituted with TLR7 and TLR8 but not non-transfected 293cells (FIG. 12). During analysis of individual ribonucleoside vanadylcomplexes, it was unexpectedly determined that a mixture of theribonucleosides A, U, C, and G or the single ribonucleoside G waseffective in the absence of vanadate at stimulating PBMC to produce TNFand TLR7 or TLR8 to activate NF-kB (FIG. 12).

FIG. 12 depicts the responsiveness of human TLR7 and human TLR8 toribonucleosides. It was determined that the response by human PBMC toRNA or RVC was mediated by TLR7 or TLR8 and further that the responsecould be driven by ribonucleosides only. Human 293 cells were eithermock-transfected or transfected with human TLR7 or human TLR8 andmonitored for responsiveness to ribonucleosides. The open reading framesof human TLR7 (hTLR7) and human TLR8 (hTLR8) were amplified by PCR froma cDNA library of human PBMC using the following primers pairs: forTLR7, 5′-CACCTCTCATGCTCTGCTCTCTTC-3′ (SEQ ID NO:15) and5′-GCTAGACCGTTTCCTTGAACACCTG-3# (SEQ ID NO:16); and for TLR8,5′-CTGCGCTGCTGCAAGTTACGGAATG-3′ (SEQ ID NO:17) and5′-GCGCGAAATCATGACTTAACGTCAG-3′ (SEQ ID NO:18). The sequence informationfor primer selection was obtained from Genbank accession numbersAF240467 and AF245703. All full-length TLR fragments were cloned intopGEM-T Easy vector (Promega, Mannheim, Germany), excised with NotI,cloned into the expression vector pcDNA 3.1 (−) (Invitrogen, Karlsruhe,Germany) and sequenced. Sequences of the coding region of hTLR7 andhTLR8 correspond to the accession numbers AF240467 (SEQ ID NO:25) andAF245703, respectively (SEQ ID NO:29).

For monitoring transient NF-κB activation, 3×10⁶ 293 HEK cells (ATCC,VA, USA) were electroporated at 200 volt and 960 μF with 1 μg TLRexpression plasmid, 20 ng NF-κB luciferase reporter-plasmid and 14 μg ofpcDNA3.1 (−) plasmid as carrier in 400 μl RPMI medium supplemented with25% fetal bovine serum (FCS). Cells were seeded at 10⁵ cells per welland after over night culture stimulated with R-848 (denoted in FIG. 12as X; commercially synthesized by GLSynthesis Inc., Worcester, Mass.,USA), RVCs or ribonucleosides for a further 7 hours. Stimulated cellswere lysed using reporter lysis buffer (Promega, Mannheim, Germany), andlysate was assayed for luciferase activity using a Berthold luminometer(Wildbad, Germany).

As depicted in FIG. 12, TLR7 transfectants responded to R-848, RVCs, amixture of ribonucleosides (A, G, C, U at 0.5 mM) and the ribonucleosideguanosine. Likewise TLR8 showed a similar response pattern.

Example 16 Responsiveness of TLR7 and TLR8 to Mixtures of TwoRibonucleosides

FIG. 13 depicts the responsiveness of TLR7 and TLR8 to mixtures of tworibonucleosides. In an experiment conducted as in FIG. 11 it wasdetermined that TLR 8 responded best to a combination of theribonucleosides G and U, however, TLR7 responded best to G alone.Additionally it can be seen that a minor response was given by acombination of C and U. These data show that ribonucleosides of theproper composition serve as ligands for TLR7 and TLR8. The nonspecificstimulus of TPA served as a control only. X denotes R-848.

Example 17 Human PBMC Respond to a Mixture of the Ribonucleosides G andU

FIG. 14 depicts the response of human PBMC to a mixture of theribonucleosides G and U. It can be appreciated that the ribonucleosidesG and U act synergistically to induce TNF from human PBMC. In thisexample the ratio of G:U of 1:10 was optimal.

Example 18 Human PBMC Respond to G,U-Rich Oligoribonucleotides

FIG. 15 depicts how human PBMC respond to RNA G,U-rich oligonucleotides.Both RNA and DNA oligonucleotides 5′-GUUGUGGUUGUGGUUGUG-3′ (SEQ ID NOs:1and 19) were tested at 30 μM on human PBMC and TNF was monitored. HumanPBMC were responsive to G,U-rich RNA oligonucleotides and not G,U-richDNA oligonucleotides.

Example 19 Human PBMC Respond to Oxidized RNA

FIG. 16 depicts the response of human PBMC to oxidized RNA. Ribosomal16S RNA was isolated from E. coli and subjected to chemical oxidation.The treatments were (mod A) 0.2 mM ascorbic acid plus 0.2 mM CuCl₂ for30 min at 37° C. or (mod B) 0.2 mM ascorbic acid plus 0.02 mM CuCl₂ for30 min at 37° C. This treatment induces oxidation at the 8 position ofguanosine and also induces strand breaks 3′ of the modified guanosine.It was shown that ribosomal RNA induced TNF production from human PBMC.It was also evident that oxidation of ribosomal RNA greatly potentiatesthe response.

Example 20 Human TLR7 Responds to Oxidized Guanosine Ribonucleoside

FIG. 17 depicts human TLR7 and TLR8 responses to the oxidized guanosineribonucleoside. Cells mock-transfected or transfected with human TLR 7or human TLR8, as in Example 14, were tested for responsiveness to7-allyl-8-oxoguanosine (loxoribine) at 1 mM. It can be clearly shownthat human TLR7 is responsive to 7-allyl-8-oxoguanosine. Thus it appearsthat a ligand for TLR 7 is oxidized nucleic acids.

Example 21 Human TLR7 Responds to Other Modified GuanosineRibonucleoside

FIG. 18 depicts human TLR7 responses to the other modified guanosineribonucleoside. Cells transfected with human TLR7, as in Example 14,were tested for a dose-dependent response to 7-allyl-8-oxoguanosine(loxoribine). Additionally other modified guanosines were tested. It canbe clearly shown that human TLR 7 was responsive to7-allyl-8-oxoguanosine in a dose-dependent manor. As shown above, humanTLR7 was responsive to guanosine; however FIG. 18 also shows that humanTLR7 responded mildly to the deoxy form of guanosine as well as to8-bromo-guanosine.

Example 22 Distribution of Human TLRs

FIG. 19 depicts the distribution of human TLR1-TLR9. Various purifiedhuman immune cells were screened by PCR for TLR1 through 9 expression.It was shown that human lymphoid CD123+ dendritic cells (DC) werestrongly positive for TLR9 and TLR7 while weaker for TLR8. The conversewas shown however for myeloid CD11c+ DC. This is very relevant becausethe two types of DC have very different functions in the immune system.Significantly, FIG. 19 also shows that human neutrophils were stronglypositive for human TLR8 while very weak for TLR9 and negative for TLR7.This is also relevant because neutrophils are very often the first cellsto engage infectious pathogens and thus believed to initiate responses.

Example 23

HEK-293 cell were stably transfected with human TLR7 or human TLR8.Additionally, the cells were stably transfected with NF-κB-luciferasereporter construct. The cells were titrated with varing amounts of RNAoligonucleotides and cultured for 16 h. Luciferase activity was measuredby standard methods and normalizied versus mock-stimulatedtransfectants. Luciferase activity measured for the mock-stimulatedtransfectant was set to a value of 1-fold NF-κB induction. Results areshown in FIG. 20, where old NF-κB induced by the stimulating RNAoligonucleotide is plotted versus the concentration of testribonucleotide. Stimulation with GUGUGUGU is shown for human TLR8.Stimulation with GUAGUCAC is shown for human TLR7 and human TLR8.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

We claim:
 1. A method of inducing an immune response in a subject to anantigen, the method comprising: administering a composition comprisingan isolated single-stranded RNA (ssRNA) G,U-rich oligoribonucleotide5-40 nucleotides long having at least 80 percent G and U to a subjectvia a route of administration selected from the group consisting ofmucosal, oral, intranasal, sublingual, ocular, vaginal, rectal, buccal,and by inhalation, wherein the G,U-rich oligoribonucleotide is free ofCpG dinucleotide, wherein the administration requires cationic lipids,wherein the ssRNA is a ligand of Toll-like receptor 7 (TLR7) or TLR8,wherein the composition further comprises an antigen, and wherein thecomposition comprises an effective amount of the ssRNA G,U-richoligoribonucleotide to induce an immune response to the antigen.
 2. Themethod of claim 1, wherein TLR7- mediated activation by the ssRNAG,U-rich oligoribonucleotide leads to IFN-α secretion from plasmacytoidprecursor dendritic cells (pDCs) and TLR8- mediated activation by thessRNA G,U-rich oligoribonucleotide leads to TNF-α secretion frommonocytes.
 3. The method of claim 1, wherein the antigen is selectedfrom the group consisting of an allergen, an antigen of a bacterium, anantigen of a virus, an antigen of a fungus, an antigen of a parasite,and a cancer antigen.
 4. The method of claim 1, wherein the antigencomprises a peptide or a polypeptide.
 5. The method of claim 1, whereinthe antigen comprises a cell.
 6. The method of claim 1, wherein thecomposition is administered to the subject more than once.
 7. The methodof claim 1, wherein the immune response comprises a Th1 immune response.8. The method of claim 1, wherein the ssRNA G,U-rich oligoribonucleotideis a ligand of both TLR7 and TLR8.